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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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-1b-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 CD81 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 iso- lated 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- . 1 , 1 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 3 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-homo- phenylalanine-vinyl sulfone methyl; ZVAD, z-Val-Ala-DL-Asp; DiOC6, 3,39-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 Dc late Ag; m, mitochondrial membrane potential; DFF, DNA fragmentation factor. anti-mouse Ig-coated Dynabeads (Dynal AS, Oslo, Norway). The resulting Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 2479 Dc FIGURE 1. PS exposure, m, and DNA strand breaks in GC B lymphocytes upon contact with Dc 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 .98% CD381 cells and ,2% Phosphatidyl serine (PS) exposure was determined using annexin V- CD391 and surface IgD1 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%.