Both Caspase and Cathepsin Activity Germinal Center B Cell Apoptosis

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Germinal Center B Cell Apoptosis Requires Both Caspase and Cathepsin Activity Marco van Eijk and Cornelis de Groot This information is current as J Immunol 1999; 163:2478-2482; ; of September 29, 2021. http://www.jimmunol.org/content/163/5/2478 Downloaded from References This article cites 35 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/163/5/2478.full#ref-list-1

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Germinal Center B Cell Apoptosis Requires Both Caspase and Cathepsin Activity

Marco van Eijk and Cornelis de Groot1

Follicular dendritic cells (FDCs) select B cells during germinal center (GC) reactions. The B cells that are able to bind to the FDCs receive a signal that leads to the termination of endonuclease activity in the nuclei of those B cells. This signal must be in addition to the signals transferred through the cross-linkage of the B cell receptors and signals resulting from the interactions of the adhesion molecules lymphocyte function-associated Ag-1 and very late Ag-4 with ICAM-1 and VCAM-1, respectively. In this report, we present evidence that the FDCs silence all apoptotic processes in GC B lymphocytes and additionally switch off pre-present endonuclease activity. We also show that GC B cell apoptosis requires cathepsin activity downstream of caspase-3. This cathepsin activity is directly connected to endonuclease activity and therefore may be an interesting target for the antiapop-

totic factors produced by FDCs. The Journal of Immunology, 1999, 163: 2478–2482. Downloaded from

erminal centers (GCs)2 are specialized microenviron- To gain insight into the specific rescue mechanism of FDCs, it ments in lymphoid follicles of secondary lymphoid or- is necessary to know what routes are used to trigger apoptosis in G gans. Here, B lymphocytes undergo affinity maturation GC B cells. Cysteine proteases fulfill crucial roles in apoptosis. For of their B cell receptors (BCR) and Ig isotype-switch, resulting in instance, the family of IL-1␤-converting enzyme-like proteases the formation of memory B cells (1–3). During a GC reaction, B (now called caspases) (14–16) forms an important cascade that http://www.jimmunol.org/ cells are selected and their Ag specificity is checked at different links triggering signals such as Fas ligation to the final activation levels (4). Antiapoptotic signals provided by follicular dendritic of DNA fragmentation (17–19). This cascade is highly redundant; cells (FDCs) are crucial in this selection process. Native Ags are however, in general, the activation of various members of the presented to GC B cells in the immune complexes present on caspase family may lead to the activation of caspase-3, resulting in FDCs, allowing B lymphocytes with high affinity BCRs to bind. cleavage of various substrates that are crucial in the execution As a result of this highly competitive binding, the programmed cell phase of apoptosis. In addition, it was shown recently that mem- death of the GC B cells is cancelled in the binding cells only (5, 6). bers of the papain family of cysteine proteases may be involved in FDCs protect the attached B lymphocytes in such a way that en- apoptotic processes as well. For example, calpains are involved in

donuclease, which is pre-present in the nuclei of GC B cells, is the upstream regulation of thymocyte apoptosis (20, 21) and re- by guest on September 29, 2021 switched off within a few hours (7). cently, cathepsin W, also called lymphopain, was found in CD8ϩ The precise mechanism of this action is largely unknown. BCR T lymphocytes and NK cells, suggesting a role in the apoptosis cross-linkage with Ag in the immune complexes on FDCs is an pathway that is used for target cell killing (22, 23). important prerequisite; however, interactions between the adhesion In the present paper, we have addressed the role of FDCs on GC molecules ICAM-1 (CD54) and VCAM-1 (CD106) with lympho- B cell apoptosis, and especially the enzymes involved in the reg- cyte function-associated Ag-1 (LFA-1) and very late Ag-4 ulation of endonuclease activity. Our experiments indicate that (VLA-4) (CD49d), respectively, also play a role in the intimate both caspase and cathepsin activity are required in the apoptotic contact between B lymphocytes and FDCs (8–10). Thus far, the cascade of GC B cells. Furthermore, we show that the cathepsin apoptosis of GC B cells could be postponed by cross-linkage of activity acts downstream of caspases and is probably the last pro- LFA-1, VLA-4, CD21, CD40, BCR, or CD40 and BCR; however, teolytic step involved in the activation of DNA fragmentation. none of these signals could switch off endonuclease activity in GC FDCs, therefore, may act on endonuclease activity directly be- B cells (5, 7, 9–13). cause that is the only apoptotic parameter present in freshly isolated GC B cells (F-B) that is switched off.

Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Materials and Methods Received for publication April 13, 1999. Accepted for publication June 11, 1999. Isolation of GC B cells from human tonsils The costs of publication of this article were defrayed in part by the payment of page B lymphocytes were isolated from tonsils according to the method of Lind- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. hout et al. (7). Brieﬂy, tonsillar cell suspensions were depleted of T cells using 2-aminoethylisothiouroniumhydrobromide (Sigma, St. Louis, MO) 1 Address correspondence and reprint requests to Dr. Cornelis de Groot, Department -treated SRBCs (24) followed by density centrifugation on a Lymphoprep of Cell Biology and Histology, Cellular Immunology Group, Academic Medical Cen- (1077 mg/ml; Nycomed, Oslo, Norway) to remove rosetted cells. The ﬁnal ter, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Nether- Ͼ ϩ Ͻ ϩ lands. E-mail address: [email protected] cell population contained 98% CD20 cells (B cells) and 2% CD3 cells (T cells). This B cell suspension was centrifuged (15 min, 1200 ϫ g, 2 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic 4°C) on a Percoll gradient (Pharmacia, Uppsala, Sweden) consisting of cell; F-B, freshly isolated B cells; N-B, nonclustered B cells; C-B, B cells clustered three layers (1043, 1067, and 1077 mg/ml). Cells at the 1043/1067 inter- to FDCs; BCR, B cell receptor; ZPP, Z-phenyl-phenyl-CHN2; LHVS, leucine-homophenylalanine-vinyl sulfone methyl; ZVAD, z-Val-Ala-DL-Asp; DiOC6, 3,3Ј-di- face were collected and incubated with Abs against surface IgD (MAS hexyloxacarbocyanine iodide; PI, propidium iodide; PS, phosphatidyl serine; PARP, 590p, Harlan Sera-Lab, Loughborough, U.K.) and anti-CD39 (AC2, Im- poly(ADP-ribose)-polymerase; LFA, lymphocyte function-associated Ag; VLA, very munotech, Marseilles, France). Labeled cells were depleted using sheep ⌬␺ late Ag; m, mitochondrial membrane potential; DFF, DNA fragmentation factor. anti-mouse Ig-coated Dynabeads (Dynal AS, Oslo, Norway). The resulting

⌬␺ FIGURE 1. PS exposure, m, and DNA strand breaks in GC B lymphocytes upon contact with ⌬␺ FDCs. a, PS exposure; b, m; c, DNA strand breaks. Apoptotic parameters were determined in F-B, N-B, and C-B. In a–c, an increase is observed in the N-B fraction. One representative example of at least three experiments is shown. Downloaded from

puriﬁed GC B cell fractions consisted of Ͼ98% CD38ϩ cells and Ͻ2% Phosphatidyl serine (PS) exposure was determined using annexin V- CD39ϩ and surface IgDϩ cells. FITC (Bender Medsystems Diagnostics, Vienna, Austria) in combination with PI. Cells were labeled with annexin V-FITC for 30 min on ice,

Isolation of FDCs washed, and taken up in medium containing PI. http://www.jimmunol.org/ Caspase-3 activity was measured using the ApoAlert CPP32 Fluores- FDCs were isolated from tonsils as described by Parmentier et al. (25). cent Assay Kit (Clontech, Palo Alto, CA). Briefly, before or after the in- Tonsils were cut into pieces and treated with a collagenase (200 U/ml duction of apoptosis, cells were lysed and taken up in reaction buffer. After collagenase IV, Worthington Biochemical, Lakewood, NJ)/DNase (10 addition of the 7-amino-4-trifluoromethyl coumarin-peptide substrate con- U/ml DNase I, Boehringer Mannheim, Mannheim, Germany) solution in jugate and subsequent incubation at 37°C, the enzyme activity was mon- IMDM (Life Technologies, Paisley, U.K.), followed by density sedimen- itored in a JASCO FP-750 spectrofluorometer (B&L Systems, Maarssen, tation on a cold discontinuous BSA (Path-o-cyte 4, bovine albumin, In- The Netherlands). struchemie, Hilversum, The Netherlands) gradient in HBSS (Life Tech- Poly(ADP-ribose)-polymerase (PARP) fragmentation was assessed by nologies), consisting of layers of 1.5, 2.5, and 5%. The cells at the 2.5–5% Western blotting using a rabbit polyclonal Ab (Boehringer Mannheim). interface were harvested and washed in IMDM. These FDC-enriched frac- Blots were stained with HRP-conjugated goat anti-rabbit IgG (Nordic Im- tions were used in the experiments. by guest on September 29, 2021 munological Laboratories, Tilburg, The Netherlands) using enhanced Cell cultures chemiluminescence Western blotting detection reagents (Amersham Inter- national, Little Chalfont, U.K.). All standard media used (IMDM, HBSS) contained gentamicin (90 ␮g/ml). Endonuclease activity was determined as described by Lindhout et al. B cells were cultured for4hin24-well culture plates (Costar, Cambridge, (7). Briefly, isolated nuclei were incubated at 37°C for4hinTSNbuffer MA) in IMDM supplemented with 10% FCS (HyClone, Logan, UT) in the (10 mM Tris-HCl, 200 mM sucrose, and 60 mM NaCl (pH 7.5)) followed presence or absence of the general cathepsin inhibitor E64d, the cathepsin by the separation of large and small DNA fragments; these fragments were B inhibitor CA074-Me (Scientific Marketing Associates, Barnet, U.K.), the precipitated and run on an agarose gel. cathepsin L inhibitor Z-phenyl-phenyl-CHN2 (ZPP) (Enzyme System Fluorescence analyses of DiOC6 (3) signal, DNA strand breaks, and Products, Dublin, CA), the cathepsin S inhibitor morpholine urea leucine- annexin V-FITC staining were conducted on a FACScan (Becton Dickin- homophenylalanine-vinyl sulfone methyl (LHVS) (a kind gift of Dr. Re- son, Mountain View, CA) and analyzed using WinMDi 2.7 software de- becca A.R. Bryant, Harvard Medical School, Boston, MA), and the broad veloped by Joseph Trotter (Scripps Research Institute, La Jolla, CA). range caspase inhibitor z-Val-Ala-DL-Asp (ZVAD)-fluoromethylketone (Alexis Corporation, Laüfelfingen, Switzerland). The inhibitors were dis- Results solved in DMSO (Sigma) and applied at concentrations ranging from 50 FDCs silence a variety of apoptotic signals and additionally nM to 150 ␮M as indicated. switch off endonuclease activity in GC B lymphocytes FDC-enriched cell suspensions were depleted of T cells using SRBCs and cultured in IMDM supplemented with 10% FCS. After 14 h, FDC-B Apoptotic parameters were measured and compared in F-B, in B cell clusters were separated from single B cells by 1 ϫ g sedimentation on cells clustered to FDCs (C-B), and in nonclustered B cells (N-B) IMDM with 30% FCS for 30 min at 0°C. Clusters were isolated from the that were separated from each other by 1 ϫ g sedimentation on pellet, and single B cells were harvested from the interface. Apoptotic parameters were determined as described below. IMDM with 30% FCS. Several apoptotic parameters were analyzed: PS exposure using annexin V-FITC staining (Fig. 1a), reduction of Detection of apoptotic parameters ⌬␺ m using DiOC6 (3) staining (Fig. 1b), DNA strand breaks using the ⌬␺ in situ cell death detection method (Fig. 1c), caspase-3 activity The reduction of mitochondrial membrane potential ( m) was analyzed according to the method of Zamzami et al. (26) using 3,3Ј-dihexyloxacar- by monitoring the cleavage product of the caspase-3 specific substrate bocyanine iodide (DiOC6 (3)) (Molecular Probes, Leiden, The Nether- 7-amino-4-trifluoromethyl coumarin-peptide substrate conjugate (Fig. lands) in combination with propidium iodide (PI) (Sigma). DiOC6 (3) was 2a), cleavage of PARP (Fig. 2b), and endonuclease activity in isolated applied to the cells at a concentration of 40 nM in PBS for 15 min at 37°C. The cells were washed and taken up in PBS. PI (50 ng/ml final concen- nuclei of GC B cells (Fig. 2c). tration) was added, and samples were directly analyzed. As is shown in Fig. 1, a–c, F-B showed low levels of PS ex- ⌬␺ DNA strand breaks were analyzed using the In Situ Cell Death Detec- posure, a high m, and very few DNA strand breaks. Caspase-3 tion Kit (Boehringer Mannheim) according to the manufacturer’s instruc- is inactive in these cells (Fig. 2a), which is consistent with the tions. Briefly, 1–2 ϫ 106 B cells were washed in PBS with 1% BSA, taken up in PBS and paraformaldehyde (2% final concentration), and permeabil- finding that the DNA repair enzyme PARP is present in its un- ized in 0.1% Triton X-100 and 0.1% sodium citrate. dUTP-fluorescein was cleaved 113-kDa form (Fig. 2b). These data confirm the nonapop- coupled to the DNA strand breaks using terminal transferase. totic phenotype of F-B. 2480 CATHEPSIN ACTIVITY DURING GC B CELL APOPTOSIS

FIGURE 3. Inhibition of caspase activity or cathepsin activity blocks the occurrence of DNA strand breaks in GC B cells. Puriﬁed GC B lymphocytes were incubated for4hat37°C in IMDM/10% FCS in the presence or absence of the general caspase inhibitor ZVAD (150 ␮M), the general cathepsin inhibitor E64d (60 ␮M), the cathepsin B inhibitor Downloaded from CA074-Me (20 ␮M), the cathepsin L inhibitor ZPP (10 ␮M), and the cathepsin S inhibitor LHVS (50 nM); DNA strand breaks were analyzed using an in situ cell death detection kit. Both E64d and ZVAD were able to inhibit DNA strand breaks, whereas CA074-Me, ZPP, and LHVS could not. Data are expressed as a percentage of the inhibition of DNA strand break formation. Positive and negative controls were 0% and 100% cells Ϯ with DNA strand breaks, respectively. Results are expressed as mean http://www.jimmunol.org/ SD (n ϭ 3).

Apoptosis of GC B cells involves both caspase and cathepsin activity To investigate whether caspases and other cysteine proteases, such as cathepsins, were involved in the apoptotic machinery of GC B

lymphocytes, puriﬁed GC B cells were cultured in the presence or by guest on September 29, 2021 absence of various cathepsin inhibitors and the general caspase FIGURE 2. Caspase-3, PARP, and DNA fragmentation in GC B lym- inhibitor ZVAD (Fig. 3). DNA strand breaks were analyzed after phocytes upon contact with FDCs. a, caspase-3 activity; b, PARP cleavage; 4 h using the in situ cell death detection assay. Strikingly, DNA c, endonuclease activity in isolated nuclei. Apoptotic parameters were de- strand breaks were blocked not only with the caspase inhibitor termined in F-B, N-B, and C-B. Caspase-3 activity is expressed as a per- ZVAD but also with the general cathepsin inhibitor E64d. How- centage of the values found in the F-B fraction. In a–c, an increase is ever, this was not observed when speciﬁc inhibitors for cathepsin observed in the N-B fraction. In c, the latent endonuclease activity in the B (CA074-Me), cathepsin L (ZPP), or cathepsin S (LHVS) were F-B fraction is switched off in the C-B fraction. One representative exam- used. These data imply that both caspase and cathepsin activities ple of at least three experiments is shown with respect to PARP cleavage are required for the occurrence of DNA strand breaks in GC B and endonuclease activity (b and c). The results in a are expressed as cells, and that caspase(s) and cathepsin(s) must act in a sequential mean Ϯ SD (n ϭ 3). manner.

Cathepsins act downstream of caspase-3 on endonuclease After 14 h however, the single B cell fraction (N-B) showed a activity in GC B lymphocytes ⌬␺ largely increased level of PS exposure, reduced m, and in- To further investigate at which level the cathepsins were involved creased numbers of DNA strand breaks (Fig. 1, a–c). Moreover, in the apoptotic cascade, the effects of the cathepsin inhibitor E64d caspase-3 activity could be demonstrated at this timepoint (Fig. and the caspase inhibitor ZVAD on different apoptotic processes ⌬␺ 2a). This is in agreement with the ﬁnding that PARP was now were measured, including PS exposure, reduction of m, present as an inactivated 89-kDa fragment (Fig. 2b). caspase-3 activity, cleavage of PARP, and DNA strand breaks. As ⌬␺ By contrast, the C-B showed levels of PS exposure, m, DNA shown in Fig. 4a, PS exposure was inhibited by ZVAD but not by strand breaks, PARP cleavage, and caspase-3 activity that were E64d. Also, ZVAD inhibited mitochondrial damage (Fig. 4b), similar to the F-B. caspase-3 activity (Fig. 4c), and processing of the caspase-3 sub- Interestingly, endonuclease activity could be demonstrated in strate PARP (Fig. 4d). E64d did not block any of these processes, the nuclei of both F-B (Fig. 2c, lane 2) and the single B cell indicating that cathepsin activity could not reside upstream of fraction (N-B) (Fig. 2c, lane 3). In contrast, endonuclease activity caspase activity. Clear inhibition by E64d was only found when (Fig. 2c, lane 4) could not be demonstrated anymore in isolated DNA strand breaks were studied (Fig. 4e). These data demonstrate nuclei of C-B. that a thus far unidentiﬁed cathepsin activity is involved in one of the These data clearly show that FDCs keep the entire apoptotic ma- very downstream steps of the apoptotic machinery, and that this ac- chinery in GC B cells silent and furthermore switch off the latent tivity is essential for the execution of DNA fragmentation. Conse- endonuclease activity in the nuclei of the GC B lymphocytes. quently, cathepsin activity must be downstream of caspase-3 activity. The Journal of Immunology 2481

FIGURE 4. Cathepsin activity is required for the appearance of DNA strand breaks but not for upstream caspase-dependent activities. Puriﬁed GC B cells were incubated for 4 h at 37°C in IMDM/10% FCS with or without E64d or ZVAD; a variety of apoptotic Downloaded from parameters were determined, including PS exposure (a), mitochondrial inactivation (b), relative caspase-3 activity (c), PARP cleavage (d), and the occurrence of DNA strand breaks (e). Cathepsin activity is essential for DNA strand break formation only, and consequently acts downstream of caspase-3. The http://www.jimmunol.org/ data shown for PARP cleavage in d are from one representative example of at least four experiments with different donors. Control indicates GC B cells incubated for4hat 37°C without inhibitors. Results shown in a, b, c, and e are expressed as the mean Ϯ SD (n ϭ 4). by guest on September 29, 2021

Discussion survival has been shown in many studies in which B cell binding was impaired and/or apoptosis was delayed or inhibited by Abs to We have shown previously that the binding of GC B cells to FDCs the BCR, LFA-1, VLA-4, ICAM-1, or VCAM-1 (5, 8–10). in vitro results in the silencing of endonuclease activity in the B However, the cross-linkage of these molecules, alone or in cell nuclei within 4 h (7). Recently, we have found that as few as 1–2 h may be sufficient for this process (data not shown). combination, could not entirely switch off the apoptotic capacity of Thus far, the enzymatic activities that are operational during GC the B cell (i.e., it failed to block the latent endonuclease in their B cell apoptosis as well as the mechanisms used by FDCs to rescue nuclei) (7). At this stage, the rescue process is essentially CD40 the binding B lymphocytes are poorly understood. Many factors independent; although CD40 ligation postpones GC B cell death, contribute to the rescue process, but none of these, either alone or it cannot eliminate the endonuclease activity (7). Consequently, in concert, seem to explain the rapid switch-off of nuclear endo- some additional, unique signal given by FDCs must be implicated nuclease in GC B cells by FDCs. Obviously, BCR cross-linkage for the definitive switch-off of this endonuclease. with Ag present in immune complexes on the surface of FDCs The present paper confirms and extends previous studies dem- must be implicated as a first step in the binding process in vivo. onstrating this unique function of FDCs (7). In the absence of Subsequent interactions of adhesion molecules, including LFA-1/ FDCs in vitro, GC B lymphocytes show several different aspects of ICAM-1 and VLA-4/VCAM-1, will establish firm contact between apoptosis, including PS exposure, mitochondrial inactivation, FDC and GC B cells. The relevance of these interactions for B cell caspase-3 activity, PARP cleavage, and DNA fragmentation. All 2482 CATHEPSIN ACTIVITY DURING GC B CELL APOPTOSIS of these processes are cancelled upon binding to FDCs. Remark- 7. Lindhout, E., A. Lakeman, and C. de Groot. 1995. Follicular dendritic cells inhibit ably, FDCs prevent the up-regulation of multiple, caspase-related apoptosis in human B lymphocytes by rapid and irreversible blockade of preexisting endonuclease. J. Exp. Med. 181:1985. events. In addition, they eliminate the latent endonuclease activity 8. Hedman, H., and E. Lundgren. 1992. Regulation of LFA-1 avidity in human B cells: that is present in the nuclei of freshly isolated B lymphocytes. requirements for dephosphorylation events for high avidity ICAM-1 binding. J. Im- We found that caspase-3 activity is absent in both freshly iso- munol. 149:2295. 9. Koopman, G., H. K. Parmentier, H. J. Schuurman, W. Newman, C. J. L. M. Meijer, and lated and FDC-bound GC B cells. Concordantly, PARP is found in S. T. Pals. 1991. Adhesion of human B cells to follicular dendritic cells involves both the its intact form only. These data are in agreement with our previous lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late experiments showing that GC B lymphocytes are resistant to Fas- antigen 4/vascular cell adhesion molecule 1 pathways. J. Exp. Med. 173:1297. 10. Koopman, G., R. M. Keehnen, E. Lindhout, W. Newman, Y. Shimizu, mediated apoptosis as long as they remain in contact with FDCs G. A. van Seventer, C. de Groot, and S. T. Pals. 1994. Adhesion through the LFA-1 (27). The reason for this resistance is still unclear, but it may be (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) path- speculated that inhibitory proteins such as FLICE-inhibitory pro- ways prevents apoptosis of germinal center B cells. J. Immunol. 152:3760. 11. Valentine, M. A., and K. A. Licciardi. 1992. Rescue from anti-IgM-induced protein or Fas apoptosis inhibitory molecule (28, 29) are instrumental grammed cell death by the B cell surface proteins CD20 and CD40. Eur. J. Immunol. in keeping the caspase route silent. Detachment of GC B cells from 22:3141. 12. Lederman, S., M. J. Yellin, A. M. Cleary, A. Pernis, G. Inghirami, L. E. Cohn, their natural counterstructures may lead to a reactivation of the L. R. Covey, J. J. Lee, P. Rothman, and L. Chess. 1994. T-BAM/CD40L on Th caspase route, resulting in the execution of apoptosis. Recently, it lymphocytes augments lymphokine-induced B cell Ig isotype switch recombination was reported that a caspase-activated DNase/DNA fragmentation and rescues B cells from programmed cell death. J. Immunol. 152:2163. 13. Bonnefoy, J. Y., S. Henchoz, D. Hardie, M. J. Holder, and J. Gordon. 1993. A subset factor (DFF)40 is released from its inhibitor, inhibitor of caspase- of anti-CD21 antibodies promotes the rescue of germinal center B cells from apo- activated DNase/DFF45, by cleavage through caspase-3 (17–19). ptosis. Eur. J. Immunol. 23:969.

These ﬁndings connect the caspase route directly to the execution 14. Alnemri, E. S., D. J. Livingston, D. W. Nicholson, G. Salvesen, N. A. Thornberry, Downloaded from W. W. Wong, and J. Yuan. 1996. Human ICE/CED-3 protease nomenclature. Cell of apoptosis (i.e., to DNA fragmentation). Thus far, our attempts to 87:171. demonstrate DFF45 processing in GC B cells by Western blotting 15. Salvesen, G. S., and V. M. Dixit. 1997. Caspases: intracellular signaling by proteol- have been unsuccessful (data not shown). This may mean either ysis. Cell 91:443. that a different DNase activity is used by these cells or that the 16. Nicholson, D. W., and N. A. Thornberry. 1997. Caspases: killer proteases. Trends Biochem. Sci. 22:299. activation mechanism of the DNase is different. 17. Liu, X., H. Zou, C. Slaughter, and X. Wang. 1997. DFF, a heterodimeric protein that

Here, we have shown that an additional protease (i.e., a thus far functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. http://www.jimmunol.org/ unidentified cathepsin) is part of the apoptotic cascade of GC B Cell 89:175. 18. Sakahira, H., M. Enari, and S. Nagata.1998. Cleavage of CAD inhibitor in CAD lymphocytes. Inhibition of this cathepsin with the inhibitor E64d activation and DNA degradation during apoptosis. Nature 391:96. completely prevents DNA strand breaks but leaves the caspase- 19. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, and S. Nagata.1998. dependent PS exposition and PARP cleavage untouched, demon- A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43. strating that the cathepsin acts downstream of caspase-3. 20. Squier, M. K., A. C. Miller, A. M. Malkinson, and J. J. Cohen. 1994. Calpain acti- It is tempting to speculate that blockade of this very downstream vation in apoptosis. J. Cell Physiol. 159:229. cathepsin activity may well be the target of FDC action. Natural 21. Squier, M. K., and J. J. Cohen. 1997. Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158:3690. inhibitors of cathepsins belong to the family of cystatins (30, 31), 22. Linnevers, C., S. P. Smeekens, and D. Bromme. 1997. Human cathepsin W, a pu- and it has been demonstrated that cathepsins and cystatins are in- tative cysteine protease predominantly expressed in CD8ϩ T-lymphocytes. FEBS by guest on September 29, 2021 volved in different models of apoptosis. For instance, in a model Lett. 405:253. 23. Brown, J., E. Matutes, A. Singleton, C. Price, H. Molgaard, D. Buttle, and T. Enver. for bile-salt induced apoptosis in hepatocytes, it was shown that 1998. Lymphopain, a cytotoxic T- and natural killer cell-associated cysteine protein- cathepsin B was downstream of caspase-3. Cystatin A could effi- ase. Leukemia 12:1771. ciently block apoptosis in this model (32). In addition, increased 24. Falkoff, R. M., M. Peters, and A. S. Fauci. 1982. T cell enrichment and depletion of human peripheral blood mononuclear cell preparations: unexpected findings in the study of the apoptosis was reported in the brain of the cystatin B knockout functional activities of the separated populations. J. Immunol. Methods. 50:39. mouse (33). Moreover, it was shown that cystatin A could inhibit 25. Parmentier, H. K., J. A. van der Linden, J. Krijnen, D. F. van Wichen, the virus-induced apoptosis of a carp cell line (34). L. H. P. M. Rademakers, A. C. Bloem, and H. J. Schuurman. 1991. Human follicular dendritic cells: isolation and characterization in situ and in suspension. Scand. J. Im- Interestingly, cystatin A is present in FDCs (35); it is attractive munol. 33:441. to consider the possibility that it is transported to B cells during the 26. Zamzami, N., P. Marchetti, M. Castedo, C. Zanin, J. L. Vayssiere, P. X. Petit, and intimate contact with FDCs. 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