B Lymphocytes to Enter the Cell Cycle Caspase Activity Is Required For

Caspase Activity Is Required for Stimulated B Lymphocytes to Enter the Cell Cycle N. Eric Olson, Jonathan D. Graves, Geraldine L. Shu, Elizabeth J. Ryan and Edward A. Clark This information is current as of September 30, 2021. J Immunol 2003; 170:6065-6072; ; doi: 10.4049/jimmunol.170.12.6065 http://www.jimmunol.org/content/170/12/6065 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Caspase Activity Is Required for Stimulated B Lymphocytes to Enter the Cell Cycle1

N. Eric Olson,2* Jonathan D. Graves,2† Geraldine L. Shu,2* Elizabeth J. Ryan,* and Edward A. Clark3*†

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G0) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad speciﬁcity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G1. In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was Downloaded from not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G0 B cells. These ﬁndings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle. The Journal of Immunology, 2003, 170: 6065–6072.

he mechanisms that regulate the survival and homeostatic quiescent state (7, 8). It remains unclear whether lung Kruppel-like http://www.jimmunol.org/ proliferation of mature B lymphocytes are only partially factor or SATB1 plays a similar role in B cell homeostasis, which T understood. The number of mature B cells is not influ- is regulated separately from T cell homeostasis (4). enced as significantly by daily rates of B cell production in the Another mechanism known to contribute to the homeostatic reg- bone marrow as by active mechanisms in the periphery (1). The B ulation of B cell populations is programmed cell death or apopto- cell Ag receptor complex is required for mature B cells to survive sis. The main effectors of cell death are a family of cysteine pro- (2), and as yet poorly understood B cell Ag receptor-dependent teases called caspases (reviewed in Refs. 9 and 10). The best- selective mechanisms, such as the regulation of transitional B cells characterized pathway for caspase activation is via the CD95/Fas in the spleen (3), appear to regulate B cell pool size (4). receptor. Activated CD95/Fas trimers recruit the pro form of by guest on September 30, 2021 Quiescent B cells express a distinct set of genes from either caspase-8 via the adapter protein Fas-associated death domain pro- anergic or activated B cells (5), suggesting that the decision to tein (FADD) (9, 10). The resulting oligomerization of pro- remain quiescent is an actively regulated process. A number of caspase-8 results in autoprocessing and activation. Caspase-8 then proteins with known or predicted inhibitory functions have been cleaves and activates downstream caspases such as caspase-3, -6, implicated in this process (5, 6). For example, lung Kruppel-like and -7. Once activated, caspases target cleavage of a highly spe- 4 factor and special AT-rich sequence-binding protein 1 (SATB1) cific and restricted group of cellular proteins that regulate cell mor- function as transcriptional repressors that maintain T cells in a phology, signal transduction, and survival (9). Among the known targets of caspases are a number of proteins that function to regulate the cell cycle. These include the retinoblastoma protein (Rb), Departments of *Microbiology and †Immunology, University of Washington, Seattle, WA 98195 a critical regulator of cell cycle progression (11), and the cyclin- Received for publication December 2, 2002. Accepted for publication April 10, 2003. dependent kinase inhibitors, p21Cip1/Waf1 and p27Kip1 (12). Cleavage of these proteins contributes to cell cycle arrest and 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 serves a permissive role in the induction of apoptosis. with 18 U.S.C. Section 1734 solely to indicate this fact. Several reports suggest that caspases can function to regulate 1 This work was supported by National Institutes of Health Grants GM37905 and processes besides apoptosis, including proliferation and differen- RR00166 (to E.A.C.) and GM58487 (to J.D.G.). tiation (13). One of the first hints that caspase-dependent pathways 2 N.E.O., J.D.G., and G.L.S. contributed equally to this work. may regulate proliferation came from analyses of FADD-deficient 3 Address correspondence and reprint requests to Dr. Edward A. Clark, Department of mice and mice expressing dominant-negative FADD in the T cell Microbiology, Box 357242, University of Washington, Seattle WA 98195. E-mail address: eclark@ bart.rprc.washington.edu compartment. Using both these approaches, it was shown that 4 Abbreviations used in this paper: SATB1, special AT-rich sequence-binding protein FADD is required for T cell activation and IL-2-dependent prolif- 1; Ac-DEVD-AMC, acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin; Ac-IETD- eration (14Ð16). Subsequent studies suggested a positive role for AMC, acetyl-Ile-Glu-Thr-Asp-7-amino-4-methyl coumarin; Ac-VEID-AMC, scetyl- Val-Glu-Ile-Asp-7-amino-4-methyl coumarin; cdk, cyclin-dependent kinase 4; caspases during proliferation of CD3-stimulated normal human T DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-al; DEVD-fmk, benzyloxycarbonyl (Cbz)- cells (17, 18). These studies found that caspase-8 activity is asso- Asp-Glu-Val-Asp(Ome)-fluoromethylketone; ERK, extracellular signal-regulated kinase; ciated with T cell proliferation, and that either a broad specificity FADD, Fas-associated death domain protein; IAP, inhibitor of apoptosis; IETD-fmk, benzyloxycarbonyl (Cbz)-Ile-Glu-Thr-Asp(Ome)-fluoromethylketone; MAPK, mitogen- caspase inhibitor or an inhibitor selective for caspase-8 could activated protein kinase; PARP, poly-ADP ribose polymerase; Rb, retinoblastoma pro- block proliferation. More recently, Chun et al. (19) identified a tein; RPA, RNase protection assay; VEID-fmk, benzyloxycarbonyl (Cbz)-Val-Glu-Ile- Asp(Ome)-fluoromethylketone; ZVAD-CHO, N-acetyl-Val-Ala-Asp-al; ZVAD-fmk, caspase-8 mutation in patients with autoimmune lymphoprolifera- benzyloxycarbonyl (Cbz)-Val-Ala-Asp(Ome)-fluoromethylketone; cIAP, cellular IAP. tive syndrome-related symptoms. T, B, and NK cells from these

patients exhibited defects in activation consistent with a role for content was measured. Cells with sub-G0/G1 DNA content were considered caspase-8 in lymphocyte activation. However, which caspases apoptotic. function downstream of caspase-8 and how caspases might act as Western blot analysis positive regulators of the cell cycle has not been determined. In this study we investigated the role of caspases in the activa- Human tonsillar B cells were pretreated where indicated with caspase inhibitors for 3 h before stimulation. At the indicated time, 1.5 ϫ 107 cells tion of normal quiescent human B cells. We found that both were rinsed with cold PBS and lysed in a buffer containing 1% SDS and 50 caspase-8 and -6 activities increased following B cell activation by mM Tris, pH 7.6. Samples were passed through a 25-gauge needle 10 a variety of proliferative stimuli, and that inhibitors selective for times, and then insoluble matter was removed by centrifugation. A small these caspases blocked B cell proliferation. Inhibition of caspase-6 sample of the resulting supernatant was reserved for protein determination; ␤ antagonized both mRNA and protein expression of cyclin-depen- the rest was diluted 1/1 in sample buffer containing 5% -ME and boiled for 5 min. The protein concentration of each sample was determined using dent kinase 4 (cdk4) and cyclin D, suggesting that resting B cells a bicinchoninic acid assay (Pierce, Rockford, IL) with BSA as a standard. may require caspase-6 activity to enter into the G1 phase of the cell Equal amounts of total protein were loaded onto polyacrylamide gels, elec- cycle. Neither CD40-mediated IL-6 production nor cellular inhib- trophoresed, and transferred to nitrocellulose. The blot was then immuno- itor of apoptosis 2 (cIAP2) expression was affected by caspase stained using Abs specific for cyclin D3 (Upstate Biotechnology, Lake Placid, NY), caspase-3, caspase-8 (BD PharMingen, San Diego, CA), inhibition, suggesting that caspase activity is required for cell cycle caspase-6 (MLB International, Watertown, MA), cyclin A, SATB1 (Santa entry, but not cytokine or survival programs. We propose a model Cruz Biotechnology, Santa Cruz, CA), phospho-Rb, phospho-extracellular that caspases may regulate cell cycle entry by targeting proteins signal-regulated kinase (phospho-ERK), Akt/protein kinase B (Cell Sig- that actively maintain the quiescent state, thereby activating tran- naling Technology, Beverly, MA), or p27Kip1 (BD Biosciences). Briefly, the blot was first incubated in 5% nonfat dried milk in TBS-T (10 mM Tris scription of early cell cycle genes. Downloaded from (pH 7.6), 150 mM NaCl, and 0.1% Tween 20) for 1 h. The blot was then incubated with primary Ab diluted in TBS-T buffer for 1 h. After rinsing, Materials and Methods the blot was exposed to a HRP-conjugated secondary Abs diluted in Cells and Abs TBS-T. After rinsing, blots were incubated in ECL (Amersham Pharmacia Biotech) reagent for 1 min, blotted dry, and then exposed to film until a Dense tonsillar B cells were prepared as described previously (20, 21); signal was detected. briefly, E rosette-negative (ErϪ) cells were layered over a Percoll step gradient, and, after centrifugation, cells were isolated that were Ͼ60% Caspase activity assays http://www.jimmunol.org/ Percoll density (Ͼ98% CD20ϩ, Ͼ98% in G based on acridine orange 0 Following stimulation, cells were washed once in PBS and resuspended at staining). Mouse dense B cells were purified and stimulated as previously ϫ 8 ␤ described (22). The G28-5 mAb to human CD40 (23) and the G28-8 mAb 2 10 /ml in hypotonic lysis buffer (50 mM NaCl, 40 mM -glycero- to Bgp95, recently designated CD180/RP105 (20, 24), were used alone or phosphate, 10 mM HEPES (pH 7.0), 5 mM EGTA, and 2 mM MgCl2). The together to activate resting B cells. The 1C10 mAb to mouse CD40 was lysate was then subjected to four freeze-thaw cycles before centrifugation Ј at 10,000 ϫ g for 10 min (100 mM HEPES (pH 7.5), 10% sucrose, 0.1% used to stimulate mouse B cells. In some experiments F(ab )2 of goat ␮ ␮ 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 10 mM DTT, anti-mouse chains or goat anti-human sera (Jackson ImmunoResearch ␮ Laboratories, West Grove, PA) were used to stimulate B cells. and 0.1 mg/ml OVA). Protein concentrations were determined, and 25 g of cell extract was incubated for4hat37¡C with 50 ␮M Ac-DEVD-AMC, Reagents Ac-VEID-AMC, or Ac-IETD-AMC. Assays were performed in the presence or the absence of 1 ␮M ZVAD-CHO or DEVD-CHO inhibitor as a by guest on September 30, 2021 The peptide caspase inhibitors, benzyloxycarbonyl (Cbz)-Val-Ala-As- control for broad specificity caspase activity and caspase-3 activity, re- p(Ome)-fluoromethylketone (ZVAD-fmk), N-acetyl-Val-Ala-Asp-al spectively. Protease activity was determined by monitoring the release of (ZVAD-CHO), benzyloxycarbonyl (Cbz)-Ile-Glu-Thr-Asp(Ome)-fluoro- 7-amino-4-trifluoromethyl coumarin at an excitation wavelength of 400 nm methylketone (IETD-fmk), benzyloxycarbonyl (Cbz)-Asp-Glu-Val-As- and an emission wavelength of 510 nm with the use of a CytoFluor II p(Ome)-fluoromethylketone (DEVD-fmk), N-acetyl-Asp-Glu-Val-Asp-al 96-well plate spectrofluorometer (PerSeptive Biosystems, Framingham, (DEVD-CHO), and benzyloxycarbonyl (Cbz)-Val-Glu-Ile-Asp(Ome)-flu- MA). Relative caspase activity was determined by dividing the activity oromethylketone (VEID-fmk), were purchased from Kamiya Biomedical observed at each time point by the values detected at time zero. (Seattle, WA). The caspase peptide substrates, acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (Ac-DEVD-AMC), scetyl-Val-Glu-Ile-Asp-7- Cytokine assays amino-4-methyl coumarin (Ac-VEID-AMC), and acetyl-Ile-Glu-Thr-Asp- 7-amino-4-methyl coumarin (Ac-IETD-AMC), were purchased from En- B cells were stimulated as described above. Then supernatants were re- zyme System Products (Livermore, CA). Human rIL-4 was obtained from moved at 24 h, and the concentrations of IL-6, IL-10, and IL-1 were de- R&D Systems (Minneapolis, MN). termined by immunoassays. Matched pairs of Abs were as follows: IL-10, JES3-19F1 (capture) and JES3-6B11 (detection); IL-6, MQ2-13A5 (cap- Proliferation analysis ture) and MQ2-39C3 (detection; BD PharMingen); and IL-1␤, 508A7G8 and 508A4A2 (capture) and 508A3H12 (detection; BioSource Interna- Dense tonsillar B cells were cultured at 1Ð2 ϫ 106 cells/ml in triplicate at tional, Camarillo, CA). IL-6, IL-10, and IL-1␤ were detected with ExtrA- Ј Ј 37¡C and 5% CO2 in complete RPMI medium (10% FCS). Cells were then vidin-HRP (1/1000 dilution), followed by 3,3 ,5,5 -tetramethylbenzidine stimulated with anti-CD180 (5 ␮g/ml), anti-CD40 (1 ␮g/ml), anti-␮ (5 substrate (Sigma-Aldrich, St. Louis, MO). Results were expressed as mean ␮g/ml), IL-4 (10 ng/ml), or combinations of stimuli. After 2 days cells concentrations extrapolated from a standard curve prepared with recombi- were pulsed with 0.5 ␮Ci of [3H]thymidine for 18Ð24 h (for human cells) nant cytokines (BD Biosciences), for each stimulation condition performed or 8 h (for mouse cells). Identical conditions were employed for [3H]uri- in triplicate. The limit of sensitivity of each assay was 15 pg/ml. dine incorporation. Cells were then harvested on to glass-fiber filters with a cell harvester, and radioactivity was measured in a liquid scintillation RNase protection assay (RPA) counter. In some experiments cells were pretreated for 3 h with caspase RPA were performed as previously described (25) using the RiboQuant inhibitors, z-VAD-fmk, z-IETD-fmk, z-DEVD-fmk, and z-VEID-fmk, at Multiprobe RNase Protection Assay System (BD PharMingen). Total doses ranging from 5Ð80 ␮M. As a control for vehicle, cells were treated Ϫ RNAs were extracted from Er -dense tonsillar B cells that were first pre- with DMSO at the same concentration as the highest dose of inhibitor (80 treated with caspase inhibitors for 3 h, then stimulated with CD40 plus ␮M). CD180 mAb for 24 h using TRIzol reagent (Life Technologies, Grand Cell death analysis Island, NY). RPAs were then performed according to the manufacturer’s instructions, using a RiboQuant RNase protection kit and hCY-1 and Human tonsillar B cells (1 ϫ 106 cells) were rinsed with cold PBS and then hCC-1 template sets (BD PharMingen). Briefly, probes from the template resuspended in 1 ml of PBS containing 0.1% Triton X-100, 0.1 mM EDTA, sets were labeled with [␥-32P]UTP using T7 RNA polymerase and condi- and 10 U RNase A. After 25 min at room temperature, 100 ␮l of propidium tions supplied by the manufacturer. Five micrograms of the sample RNAs iodide (PI) solution (0.5 ␮g/ml) was added, and cells were incubated for were resuspended in 8 ␮l hybridization buffer, followed by the addition of another 5 min. Cells were then analyzed on a FACScan analyzer (BD 2 ␮lof32P-labeled probe (2Ð4 ϫ 105 cpm/␮l). Following the addition of Biosciences, Franklin Lakes, MJ) using the CellQuest program, and DNA the reagents, the RNA samples were then quickly denatured at 90¡C and The Journal of Immunology 6067 then allowed to anneal at 56¡C for 12Ð16 h. The samples were then treated whether caspase-8 was cleaved upon stimulation of normal B cells, with RNase A and RNase T1 mixture following the protocol described in Western blot analyses for caspase-8 was performed 48 h after stim- the kit. The samples were resolved by a 5% acrylamide sequencing gel, ulation using an Ab that recognizes the pro form as well as the which was prepared in 1ϫ TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.3). The gels were dried and analyzed by autoradiography and cleaved activated form of caspase-8. Cleaved caspase-8 was not densitometry using National Institutes of Health Image. Values were nor- detectable in the unstimulated cells and was only detected in cells malized relative to GAPDH. stimulated via CD40 alone or via CD40 in combination with CD180, surface IgM, or IL-4 signals (Fig. 1B). In subsequent ex- Results periments we stimulated G B cells with anti-CD40 plus anti- Stimulation of human G B cells increases proliferation and 0 0 CD180, since this combination consistently induced the most pro- caspase activity and decreases apoptosis liferation. Stimulation of B cells with CD40 ligand, rather than In initial experiments we compared various signals for their ability anti-CD40, in combination with anti-CD180 gave similar levels of to stimulate human dense G0 tonsillar B cells to enter the cell cycle proliferation and caspase cleavage (data not shown). These results and proliferate (Fig. 1A). The B cells used, as previously reported show that the anti-CD180 and anti-IgM combinations, which re- Ͼ ϩ Ͼ (20, 21), were 98% CD20 and 98Ð99% in G0. These G0 B sulted in the highest level of proliferation and the lowest level of cells, when left unstimulated for 3 days in culture, exhibited little apoptosis, led to caspase-8 cleavage. or no proliferation. After 3 days in culture the cells were 12Ð15% To determine whether caspase activity was also increased by annexin V positive, reflecting a basal level of apoptosis in the proliferative stimuli, we performed caspase activity assays on ly- unstimulated normal cells (Fig. 1B). Stimulation with Abs to sur- sates prepared from B cells that were either unstimulated or stim- face IgM, CD40, or CD180 (RP105/Bgp95) or with rIL-4 in- Downloaded from ulated via CD40/CD180 for 12Ð48 h. For these experiments we creased B cell proliferation at 72 h 4- to 5-fold as determined by used the peptide reporter substrates DEVD-AMC, VEID-AMC, thymidine incorporation (Fig. 1A). Combining anti-CD40 with and IETD-AMC, which are preferred substrates for caspase-3, -6, other signals (anti-CD180, anti-IgM, or IL-4) led to a significant and -8, respectively. These substrates have been widely used to increase in proliferation relative to that induced by single stimuli assess caspase activity. All peptide cleavage activity was com- (Fig. 1A).

pletely inhibited by the broad specificity caspase inhibitor ZVAD- http://www.jimmunol.org/ Previous studies suggested that caspase-8 was cleaved and activated in stimulated normal human T cells (17, 18). To determine CHO and therefore attributable to caspases. In unstimulated B cells, DEVDase (caspase-3-like) activity was detectable at all times measured and increased over the time course of the assay (Fig. 2A). This activity was completely blocked by 1 ␮M DEVD- CHO, supporting the idea that caspase-3 was probably responsible (data not shown). To exclude contaminating caspase-3 activity, assays for caspase-6 and -8 were performed in the presence of 1 ␮M DEVD-CHO. In contrast to DEVDase activity, little or no VEIDase (caspase-6-like) activity or IETDase (caspase-8-like) ac- by guest on September 30, 2021 tivity was detectable in unstimulated cells at any time point (Fig. 2, B and C). Stimulation via CD40/CD180 led to decreased DEV- Dase activity by 24 and 48 h, but increased VEIDase and IETDase activity (Fig. 2). Cleavage of VEID-AMC was detected as early as 12 h after stimulation and peaked at 24 h, while IETD-AMC cleavage was detected first at 24 h and peaked 48 h after stimulation (Fig. 2, B and C). These data are consistent with an increase in caspase-8 and -6, but a decrease in caspase-3, activity following the proliferative stimulation of normal human B cells. We next performed Western blotting for caspase-3 and -6 in cells stimulated for 24 h with CD40/CD180. Cleavage of caspase-3, indicative of activation, was observed in unstimulated B cells, but not in B cells stimulated with anti-CD40/anti-CD180 (Fig. 3A, upper panel). In contrast, significant caspase-6 cleavage was only detected in stimulated B cells (Fig. 3A, lower panel). To gain further insight into the activation state of caspase-3 and -6, we examined the cleavage state of endogenous substrates for either caspase-3 or caspase-6 in unstimulated and stimulated B cells. Ac- FIGURE 1. Effects of activation on B cell proliferation, caspase cleav- cumulated evidence suggests that poly-ADP ribose polymerase age, and apoptosis. A, Human B cells were either unstimulated or stimu- (PARP) is a preferred substrate for caspase-3, while the transcrip- lated with anti-CD40, anti-CD180, anti-IgM, or rIL-4, singly or in com- tional repressor SATB1 is primarily a substrate for caspase-6 (26, bination. After 48 h thymidine incorporation in normal human B cells was 27). While cleavage of PARP was observed in unstimulated cells, measured. Each condition was performed in triplicate, and this experiment little or no PARP cleavage was seen in B cells stimulated with is representative of more than three independent experiments. B, Western anti-CD40/anti-CD180 (Fig. 3B, upper panel). In contrast, cleav- blot analysis of cell extracts from human B cells treated as described above age of SATB1 was only seen in stimulated B cells (Fig. 3B, lower using Abs specific for human caspase-8. Proliferative stimuli increased the level of cleaved caspase-8 (active caspase species are highlighted). Apo- panel). ptosis was measured as the number of cells staining with propidium iodide We then tested the effect of cell-permeable tetrapeptide inhibi- that exhibited sub-G0 DNA content. The results presented are representa- tors, selective for either caspase-3 (DEVD-fmk) or caspase-6 tive of three independent experiments. (VEID-fmk), on PARP and SATB1 cleavage in unstimulated and 6068 CASPASES AND B CELL PROLIFERATION Downloaded from http://www.jimmunol.org/ FIGURE 2. Caspase activity following CD40/CD180 stimulation. Hu- man B cells were either unstimulated (E, ‚, and Ⅺ) or stimulated with anti-CD40/CD180 (F, Œ, and f) for the indicated time. Cell extracts were prepared and caspase activity measured using DEVD-AMC (A), VEID- AMC (B), or IETD-AMC (C) as substrate. DEVD-AMC activity was completely antagonized by the inclusion of 1 ␮M DEVD-CHO (data not shown), while data presented for VEIDase and IETDase activity were measured in the presence 1 ␮M DEVD-CHO. DEVDase (caspase-3-like) ac- FIGURE 3. Cleavage and activation of caspase-6 but not caspase-3 in tivity was decreased by anti-CD40/CD180 stimulation, while VEIDase proliferating B cells. Human B cells were either untreated or pretreated by guest on September 30, 2021 (caspase-6-like) and IETDase (caspase-8-like) activities were increased. with 80 ␮M DEVD-fmk or VEID-fmk for 3 h before stimulation with The results presented are representative of three independent experiments. anti-CD40 and anti-CD180 for 48 h. Western blot analysis was then performed on cell extracts using Abs specific for caspase-3 or caspase-6 (A), or PARP or SATB1 (B). Caspase-3 and PARP cleavage were observed in stimulated B cells. Pretreatment with DEVD-fmk blocked cleav- unstimulated cells, but not in proliferating cells. In contrast, caspase-6 and age of PARP in unstimulated B cells, but not of SATB1 in stim- SATB1 cleavage was only detected in proliferating cells. The results pre- ulated cells (Fig. 3B). Conversely, VEID-fmk inhibited SATB1 sented are representative of three independent experiments. cleavage by 70%, but only blocked PARP cleavage by 20% (Fig. 3B). These data suggest that caspase-6 activity is most likely responsible for SATB1 cleavage in stimulated B cells. Collectively, and thymidine incorporation was determined after 48 h of stimu- our Western blotting data are consistent with the data obtained by lation (Fig. 4A). VEID-fmk inhibited CD40/CD180-mediated B the peptide activity assay in suggesting that caspase-8 and -6 ac- cell proliferation over a dose range of 20Ð80 ␮M, with complete tivities are elevated in proliferating normal B cells, while inhibition seen at 80 ␮M (Fig. 4A). ZVAD-fmk was effective over caspase-3 activity is reduced. a similar concentration range as VEID-fmk, with 50% inhibition occurring between 40 and 50 ␮M. In contrast, DEVD-fmk had no Caspase activity is required for G0 B cells to proliferate effect on CD40/CD180-mediated proliferation at any concentration We next determined whether the caspase activity induced after examined. The caspase-8 inhibitor IETD-fmk achieved ϳ50% in- stimulating resting B cells is required for B cell proliferation. hibition at 80 ␮M and was consistently less effective than either Dense human B cells were pretreated for 3 h with the broad spec- ZVAD-fmk or VEID-fmk. trum caspase inhibitor ZVAD-fmk and then stimulated with anti- We next sought to determine whether the ability of caspase in- CD40 in combination with anti-CD180, anti-IgM, or IL-4. Pre- hibitors to block proliferation was restricted by stimulus, cell type, treatment with ZVAD-fmk inhibited B cell proliferation in or species. The proliferation of primary B cells in response to other response to all stimuli used (data not shown). Thus, caspase ac- combinations of stimuli, including anti-CD40 with IL-4 and anti- tivity is required for B cells to be induced to proliferate via these CD40 in addition to anti-IgM, was also blocked by caspase inhib- stimuli. We compared the dose-response of caspase-selective in- itors, indicating that the inhibitory effects of caspase inhibitors were hibitors for their ability to inhibit human B cell proliferation. not stimulus specific (data not shown). In contrast, the proliferation of Dense human B cells were pretreated for 3 h with the indicated human T cells stimulated with anti-CD3 plus anti-CD28 was not concentration of either ZVAD-fmk (a broad specificity inhibitor), inhibited by either DEVD-fmk or VEID-fmk under experimental con- IETD-fmk (selective for caspase-8), VEID-fmk (selective for ditions where B cell proliferation was significantly inhibited (Fig. 4B). caspase-6), or DEVD-fmk (selective for caspase-3). The cells were We next compared the effects of ZVAD-fmk, VEID-fmk, IETD-fmk, then stimulated with anti-CD40 in combination with anti-CD180, and DEVD-fmk on anti-CD40- or LPS-mediated proliferation in The Journal of Immunology 6069 Downloaded from

FIGURE 4. Effects of caspase inhibitors on human and mouse lymphocyte cell proliferation. A, Human B cells were either untreated (E) or pretreated with the indicated concentration of IETD-fmk (Ⅺ), DEVD-fmk (‚), ZVAD-fmk (छ), or VEID-fmk (F) before stimulation with anti-CD40 in combination with anti-CD180. B, Human tonsilar B cells or Eϩ rosette-forming T cells were either untreated (Ⅺ) or pretreated with DEVD-fmk (p) or VEID-fmk (f) before stimulation with anti-CD40 and anti-CD180 (B cells) or anti-CD3 plus anti-CD28 (T cells). C, Murine B cells were either untreated (Ⅺ) or pretreated with 80 ␮M IETD-fmk (s), DEVD-fmk (p), ZVAD-fmk (u), or VEID-fmk (f) before stimulation with either LPS or anti-CD40. D, Human B cells were http://www.jimmunol.org/ either untreated (Ⅺ) or pretreated with DEVD-fmk (p) or VEID-fmk (f) before stimulation with anti-CD40 in combination with anti-CD180. After 72 h, proliferation was measured by incorporation of [3H]thymidine, and RNA synthesis was measured by incorporation of [3H]uridine. The results presented are representative of three independent experiments. mouse splenic B cells (Fig. 4C). Both ZVAD-fmk and VEID-fmk 5, A and B). Pretreatment with VEID-fmk blocked induction of effectively inhibited LPS-induced B cell proliferation, while IETD- D-type cyclin and cdk4 mRNA by anti-CD40/CD180 almost to fmk and DEVD-fmk were less effective (Fig. 4C). The effect of basal levels (Fig. 5, A and B). However, DEVD-fmk did not inhibit ZVAD-fmk or VEID-fmk on CD40-induced splenic B cell prolifer- the induction of any of these mRNAs. In contrast to its effect on by guest on September 30, 2021 ation was reproducibly less pronounced than that on LPS-induced D-type cyclins, VEID-fmk did not influence message levels for the proliferation. It is unclear whether this reflects a difference in mouse cyclin-dependent kinase inhibitors p21 or p27 (Fig. 5, A and B). vs human B cells or differences in B cell subsets. The fact that VEID- These, findings implicate caspase-6, but not caspase-3, in induc- fmk and, to a lesser extent, IETD-fmk inhibited the proliferation of tion of D-type cyclin and cdk4 mRNA by anti-CD40/CD180. both human and murine B cells suggests that caspase-6 and -8 activity Western blot analysis using Abs specific for cell cycle proteins may be required for the proliferation of quiescent B cells. was then employed to determine the expression level of these proteins (Fig. 5C). Stimulation of quiescent B cells with anti-CD40/ Caspase activity is required for entry into the cell cycle CD180 for 48 h led to an increase in cyclin D2, cyclin D3, and Since the B cell population used in this study is highly quiescent, cdk4 protein levels. Phosphorylation of the Rb and expression of we examined the effects of caspase inhibitors on various parame- cyclin A were also significantly increased by stimulation, while ters of cell cycle entry. Two early changes that accompany cell levels of the cdk inhibitor p27Kip1 were decreased. The stimulated cycle entry include induction of RNA synthesis and an increase in accumulation of cyclin D2, cyclin D3, and cdk4 was blocked by cell size. To test whether caspase inhibitors blocked the induction preincubation with VEID-fmk, but not DEVD-fmk (Fig. 5C). of RNA synthesis we labeled cells with [3H]uridine following 72 h VEID-fmk also reduced Rb phosphorylation and blocked the de- of stimulation in the presence or the absence of caspase inhibitors crease in p27Kip1 levels observed following stimulation. The fact (Fig. 4D). While DEVD-fmk had little effect, pretreatment with that caspase-6 inhibitors block the induction of both cdk4 and D- VEID-fmk almost completely abolished the incorporation of type cyclins suggests that caspase-6 activity is required for resting 3 [ H]uridine induced by anti-CD40 and anti-CD180 (Fig. 4D, right B cells to enter the G1 phase of the cell cycle. panel). Similar results were obtained at 48 h (data not shown). In addition, analysis of forward vs side scatter FACS profiles indi- Caspase activity is not required for all CD40-dependent cated that the early increase in cell size was also blocked by activation events caspase inhibitors (data not shown). We next tested whether the caspase inhibitors selectively blocked To more precisely determine the mechanism by which caspase other signaling events associated with the stimulation of quiescent inhibitors blocked cell cycle entry of stimulated B cells, we mea- B cells. CD40 ligation has been previously shown to up-regulate sured the induction of critical cell cycle regulators. Since the levels the expression of cIAP2 (28), a protein that inhibits apoptosis by of cell cycle regulators can be influenced by both transcriptional selectively binding and inhibiting caspase-3 and caspase-7, but not and post-translational mechanisms, we measured both mRNA and caspases-6 and -8 (29). Western blot analysis of lysates from protein levels. Stimulation of quiescent B cells with anti-CD40/ CD40/CD180-stimulated B cells showed that CD40/CD180 stim- anti-CD180 for 24 h significantly increased the levels of mRNAs ulation increased cIAP2 expression. Pretreatment with ZVAD-fmk for cyclins D1 and D2 and the cyclin-dependent kinase cdk4 (Fig. had no effect on cIAP2 protein levels (Fig. 6A). We also examined 6070 CASPASES AND B CELL PROLIFERATION Downloaded from http://www.jimmunol.org/

FIGURE 6. Effects of caspase inhibitors on CD40-mediated signaling events. A, Normal tonsillar B lymphocytes were pretreated with ZVAD- by guest on September 30, 2021 fmk and then stimulated with anti-CD40/CD180 for 48 h. Expression of cIAP2 was measured by Western blotting with anti-cIAP2. B, Normal hu- FIGURE 5. Caspase-6 inhibitor blocks induction of cdk4 and D-type man tonsillar B lymphocytes were pretreated with ZVAD-fmk and then cyclins in response to proliferative stimuli. A, Human B cells were either stimulated with anti-CD40/CD180 for various times. Phosphorylation of untreated or pretreated with 80 ␮M DEVD-fmk or VEID-fmk for 3 h p42/44 ERK/MAPK was measured by Western blotting using an anti- before stimulation with anti-CD40 and anti-CD180 for 24 h. RPA for levels phospho-ERK Ab. C, Human B cells were either untreated or pre-treated of mRNA for several cell cycle proteins was performed. B, Fold induction with either 80 ␮M ZVAD-fmk, VEID-fmk, or DEVD-fmk, before stimu- values, determined by densitometry, expressed as fold induction relative to lation with anti-CD40/CD180 for 24 h. Concentrations of IL-6 and IL-10 unstimulated cells for selected mRNAs (denoted by an asterisk in A). C, in supernatants were then determined by immunoassay. Caspase inhibitors Human B cells were either untreated or pretreated with either 80 ␮M do not block induction of cIAP2, phospho-ERK or secretion of IL-6 but do DEVD-fmk or VEID-fmk for 3 h before stimulation with anti-CD40 and block secretion of IL-10. The results presented are representative of three anti-CD180 for 48 h. Western blot analysis was then performed on cell independent experiments. extracts using Abs specific for cdk4, cyclin D3, cyclin D2, p27Kip1, phospho-Rb, and cyclin A. Western blotting for p38 MAPK was performed as a control for loading. The results presented are representative of three (Fig. 6C). Treatment with ZVAD-fmk or VEID-fmk dramatically independent experiments. reduced the level of IL-10 in the supernatant of stimulated cells (Fig. 6C and data not shown). DEVD-fmk was again without effect. IL-6 levels, however, were not affected by any of the caspase the rapid phosphorylation of the ERK/MAPK and protein kinase inhibitors tested. Thus, caspase activity is selectively required for B/Akt kinases following treatment with the caspase inhibitor the induction of cell cycle genes (Figs. 4 and 5) and the B cell ZVAD-fmk. An increase in ERK phosphorylation was observed as growth factor IL-10 (31). early as 15 min after combined stimulation via CD40/CD180 (Fig. 6B), and ERK remained highly phosphorylated for at least 6 h following stimulation. Treatment with ZVAD-fmk did not affect Discussion ERK phosphorylation. ZVAD-fmk also had no effect on the phos- Proliferative stimuli activate a pattern of caspase activity that is phorylation of Akt induced by CD40/CD180 stimulation (data not distinct from apoptotic stimuli shown). Our results demonstrate that treatment of dense human B cells CD40 or CD180 stimulation up-regulates IL-6 expression in with proliferative stimuli, such as ligation of CD40 and CD180, dense human B cells (30), and IL-10 expression is regulated by leads to the selective activation of caspase-8 and caspase-6, while CD40 in normal human B cells (31). Using an ELISA to measure caspase-3 activity and apoptosis are reduced. These conclusions IL-6 and IL-10 production in supernatants, we found that CD40/ are based upon collective data including the use of peptide sub- CD180 ligation stimulated B cells to produce both IL-10 and IL-6 strates that are selective for caspases-3, -6, and -8. Proliferative The Journal of Immunology 6071 stimuli increased VEIDase and IETDase activities, while DEV- VEIDase activity was maximal 12 h after CD40/CD180 stimula- Dase activity was reduced. Although these substrates are modeled tion of quiescent B cells, caspase-8 activity did not peak until 24 h after preferred substrate sequences for the respective caspases, (Fig. 2, B and C). Furthermore, IETD-fmk was a weak and incon- their specificity is not absolute. However, in addition to this evi- sistent inhibitor of B cell proliferation (Fig. 4B and data not dence, Western blotting using Abs specific for these caspases as shown). An alternative possibility is suggested by a recent study well as for preferred substrates was also performed. Cleavage of that indicates caspase-6 functions upstream of caspase-8 in the caspase-8, caspase-6, and SATB1, but not caspase-3 or PARP, was apoptotic pathway initiated by cytochrome c (36). Thus, it is pos- observed after B cells were activated. Together these data provide sible that caspase-6 functions upstream of caspase-8 in response to strong evidence to suggest that a pattern of caspase activation dis- proliferative stimuli in B cells. In support of this hypothesis, tinct from that involved in apoptosis, involving increased IETD-fmk did not block processing and activation of caspase-6 in caspase-8 and caspase-6 activity, is induced by proliferative response to anti-CD40/CD180 (data not shown). stimuli. Although ZVAD-fmk is a potent antagonist of B cell prolifer- How might B cells activate caspase-8 and caspase-6 while pre- ation, caspase activity is not required for all activation events. For venting caspase-3 activation and apoptosis? The decrease in apo- example, caspase inhibitors had no effect on ERK/MAPK phos- ptosis may result from both the up-regulation of prosurvival fac- phorylation induced via CD40/CD180 (Fig. 6B). Since the ERK tors and the selective inactivation of effector caspases. In this pathway is required for B cell proliferation (37), this argues against respect, CD40 ligation of human B cells not only increases the the possibility that caspase inhibitors might exert a nonspecificor expression of antiapoptotic members of the Bcl-2 family, such as toxic effect blocking the activation of signaling pathways. Also, no

Bcl-2, Bcl-X, and A1 (32Ð34), but also up-regulates the expression signiﬁcant decrease in cell viability was observed following incu- Downloaded from of IAPs such as cIAP1 and cIAP2 (28). IAPs selectively bind and bation with VEID-fmk for periods up to 72 h (data not shown). inhibit caspase-3 and -7, but not caspase-6 and -8 (29). In addition, Further evidence for the speciﬁcity of caspase involvement comes cIAP2 has been shown to ubiquitinate caspase-3 and stimulate its from data showing that VEID-fmk antagonized anti- degradation by the proteasome (35). Thus, IAPs may selectively CD40/CD180-induced IL-10 secretion, but not secretion of IL-6 inactivate caspase-3 without affecting the caspases required for B (Fig. 6C). These data support the idea that caspases function down-

cells to enter the cell cycle. Interestingly, antisera specific for stream of, or parallel to, early signaling events that regulate the http://www.jimmunol.org/ caspase-3 consistently recognized several species that migrated be- proliferation, survival, and function of quiescent B cells. low the pro form in proliferating cells (Fig. 3A). The fact that these While the identity of the caspase(s) required for B cell prolif- bands are also observed upon treatment of B cells with proteasome eration cannot be definitively deduced from our present data, our inhibitors (J. D. Graves, unpublished observations) supports the results are consistent with the hypothesis that caspase-6 and/or idea that caspase-3 might be inactivated by ubiquitination in pro- another caspase, sensitive to VEID-fmk yet relatively insensitive liferating cells. The precise mechanism by which the specificity of to DEVD-fmk, is required for human B cell proliferation. Inter- caspase activation is maintained in response to proliferative stimuli estingly, a recent study involving the generation of a caspase-6- is currently under investigation. deficient chicken cell line has provided evidence that caspase-6 may be important for B cell proliferation and/or survival (38). by guest on September 30, 2021 Caspase activity is required for B cell proliferation Experiments in caspase-6-deficient mice assessing the role of Our data based upon the use of cell-permeable caspase inhibitors caspase-6 as a critical effector of B cell proliferation and matura- demonstrate that caspase activity is required for dense B cells to tion are underway. proliferate. The broad specificity caspase inhibitor ZVAD-fmk, which is an efficient inhibitor of apoptosis in many systems, Caspase-6 activity is required for entry into the cell cycle blocked B cell proliferation in response to ligation of CD40 and Pretreatment of quiescent B cells with VEID-fmk blocked induc- CD180. VEID-fmk, an inhibitor selective for caspase-6, was an tion of RNA synthesis, increases in cell size, and induction of especially effective inhibitor of proliferation, while DEVD-fmk, cyclin D2, cyclin D3, and cdk4 proteins while increasing levels of which is selective for caspase-3, was ineffective (Fig. 3B). While the cdk inhibitor p27Kip1 in response to anti-CD40/CD180 (Figs. it is important to recognize that these peptide caspase inhibitors are 4D and 5). This suggests that caspase-6 activity is required at the not absolutely specific, several lines of evidence suggest that they G0/G1 restriction point of cell cycle entry. Although p27Kip1 is exhibit a high degree of selectivity in this system. VEID-fmk com- cleaved during apoptosis, several reports also suggest that caspase pletely blocked cleavage of the caspase-6 substrate SATB1, but cleavage of p27 may serve a regulatory function during B cell only partially blocked cleavage of the caspase-3 substrate PARP, proliferation (39). Thus, one mechanism by which caspase-6 might while DEVD-fmk had no effect on SATB1 cleavage, but abolished regulate primary B cell proliferation is via cleavage of p27Kip1. that of PARP (Fig. 3B). These data also argue against differences Another potential mechanism is suggested by the fact that VEID- in permeability making a significant contribution to the differential fmk blocks the induction of mRNA encoding cdk4 and D-type effectiveness of VEID-fmk and DEVD-fmk. The fact that cyclins (Fig. 5, A and B). Further evidence to suggest that caspase caspase-6 and not caspase-3 activity is elevated in these cells pro- inhibitors block early events can be inferred from the fact that vides further evidence suggesting that VEID-fmk is likely to be VEID-fmk or ZVAD-fmk must be added within the first 4 h fol- exerting its effects via inhibition of caspase-6. lowing stimulation to block proliferation (data not shown). Ac- Based upon its apical position in the apoptotic caspase cascade cordingly, caspase-6 might target transcriptional activators or re- induced by CD95/Fas, it is tempting to conclude that caspase-8 pressors that regulate early cell cycle gene expression. Of the functions upstream of caspase-6 in regulating B cell proliferation. several defined substrates for caspase-6, SATB1 was of particular In this respect, Kennedy et al. (18) showed that caspase-8 is acti- interest. SATB1 is a matrix attachment region DNA-binding pro- vated in CD3-stimulated normal T cells and that a caspase-8 in- tein (40), is expressed in B cells (5) (Fig. 3B), is known to down- hibitor efficiently blocked CD3-mediated proliferation. Further ev- regulate gene transcription (8, 41), and after caspase cleavage loses idence for an important role for caspase-8 can be derived from a its ability to bind to chromatin (19). Activation of human B cells recent study that identified a functional deficit in caspase-8 in pa- leads to increased cleavage of SATB1 that is antagonized by tients with a lymphoproliferative disorder (19). However, while VEID-fmk. This raises the possibility that caspase-6 functions to 6072 CASPASES AND B CELL PROLIFERATION down-regulate transcriptional repressors such as SATB1 during activation caused by caspase-8 mutations lead to human immunodeficiency. Na- proliferation. ture 419:395. 20. Valentine, M. A., E. A. Clark, G. L. Shu, N. A. Norris, and J. A. Ledbetter. 1988. The homeostatic regulation of B cell populations is tightly reg- Antibody to a novel 95-kDa surface glycoprotein on human B cells induces ulated by a balance among cell death, survival, and proliferation. calcium mobilization and B cell activation. J. Immunol. 140:4071. 21. Clark, E. A., G. L. Shu, B. Luscher, K. E. Draves, J. Banchereau, J. A. Ledbetter, Our data indicate that caspases, already established as critical ef- and M. A. Valentine. 1989. Activation of human B cells: comparison of the signal fectors of cell death, also play an important role in B cell prolif- transduced by interleukin-4 to four different competence signals. J. Immunol. eration. While further analysis is required to understand the mech- 143:3873. 22. Otipoby, K. L., K. B. Andersson, K. E. Draves, A. G. Farr, S. J. Klaus, anism by which caspases contribute to these contrasting cell fates, J. D. Kerner, R. M. Perlmutter, C. L. Law, and E. A. Clark. 1996. CD22 regulates these findings raise the possibility that caspase-6 might constitute thymus-independent responses and the fate of long-lived B cells. Nature 384:634. an attractive therapeutic target for lymphoproliferative or autoim- 23. Clark, E. A., and J. A. Ledbetter. 1986. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc. mune diseases. Nat. Acad. Sci. USA 83:4494. 24. Otipoby, K. L., Y. Nagai, G. L. Shu, K. Miyake, and E. A. Clark. 2001. CD180 (RP105/Bgp95) workshop report. In Leukocyte Typing, Vol. 7. D. Y. Mason, ed. Acknowledgments Oxford University Press, Oxford, p. 120. We thank Chris Franklin for help with caspase assays, and members of the 25. Aicher, A., G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, and Clark laboratory for helpful discussions. We are grateful to the Valley E. A. Clark. 1999. Differential role for p38 mitogen-activated protein kinase in Medical Center Day Surgery for providing tonsil specimens. regulating CD40-induced gene expression in dendritic cells and B cells. J. Im- munol. 163:5786. 26. Galande, S., L. A. Dickinson, I. S. Mian, M. Sikorska, and T. Kohwi-Shigematsu. References 2001. SATB1 cleavage by caspase-6 disrupts PDZ domain-mediated dimeriza- 1. Agenes, F., M. M. Rosado, and A. A. Freitas. 1997. 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