| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The precise mechanism of this action is largely unknown. BCR cross-linkage with Ag in the immune complexes on FDCs is an important prerequisite; however, interactions between the adhesion molecules ICAM-1 (CD54) and VCAM-1 (CD106) with lymphocyte function-associated Ag-1 (LFA-1) and very late Ag-4 (VLA-4) (CD49d), respectively, also play a role in the intimate contact between B lymphocytes and FDCs (8, 9, 10). Thus far, the apoptosis of GC B cells could be postponed by cross-linkage of LFA-1, VLA-4, CD21, CD40, BCR, or CD40 and BCR; however, none of these signals could switch off endonuclease activity in GC B cells (5, 7, 9, 10, 11, 12, 13).
To gain insight into the specific rescue mechanism of FDCs, it is necessary to know what routes are used to trigger apoptosis in GC B cells. Cysteine proteases fulfill crucial roles in apoptosis. For instance, the family of IL-1ß-converting enzyme-like proteases (now called caspases) (14, 15, 16) forms an important cascade that links triggering signals such as Fas ligation to the final activation of DNA fragmentation (17, 18, 19). This cascade is highly redundant; however, in general, the activation of various members of the caspase family may lead to the activation of caspase-3, resulting in cleavage of various substrates that are crucial in the execution phase of apoptosis. In addition, it was shown recently that members of the papain family of cysteine proteases may be involved in apoptotic processes as well. For example, calpains are involved in the upstream regulation of thymocyte apoptosis (20, 21) and recently, cathepsin W, also called lymphopain, was found in CD8+ T lymphocytes and NK cells, suggesting a role in the apoptosis pathway that is used for target cell killing (22, 23).
In the present paper, we have addressed the role of FDCs on GC B cell apoptosis, and especially the enzymes involved in the regulation of endonuclease activity. Our experiments indicate that both caspase and cathepsin activity are required in the apoptotic cascade of GC B cells. Furthermore, we show that the cathepsin activity acts downstream of caspases and is probably the last proteolytic step involved in the activation of DNA fragmentation. FDCs, therefore, may act on endonuclease activity directly because that is the only apoptotic parameter present in freshly isolated GC B cells (F-B) that is switched off.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B lymphocytes were isolated from tonsils according to the method of Lindhout et al. (7). Briefly, tonsillar cell suspensions were depleted of T cells using 2-aminoethylisothiouroniumhydrobromide (Sigma, St. Louis, MO) -treated SRBCs (24) followed by density centrifugation on a Lymphoprep (1077 mg/ml; Nycomed, Oslo, Norway) to remove rosetted cells. The final cell population contained >98% CD20+ cells (B cells) and <2% CD3+ cells (T cells). This B cell suspension was centrifuged (15 min, 1200 x g, 4°C) on a Percoll gradient (Pharmacia, Uppsala, Sweden) consisting of three layers (1043, 1067, and 1077 mg/ml). Cells at the 1043/1067 interface were collected and incubated with Abs against surface IgD (MAS 590p, Harlan Sera-Lab, Loughborough, U.K.) and anti-CD39 (AC2, Immunotech, Marseilles, France). Labeled cells were depleted using sheep anti-mouse Ig-coated Dynabeads (Dynal AS, Oslo, Norway). The resulting purified GC B cell fractions consisted of >98% CD38+ cells and <2% CD39+ and surface IgD+ cells.
Isolation of FDCs
FDCs were isolated from tonsils as described by Parmentier et al. (25). Tonsils were cut into pieces and treated with a collagenase (200 U/ml collagenase IV, Worthington Biochemical, Lakewood, NJ)/DNase (10 U/ml DNase I, Boehringer Mannheim, Mannheim, Germany) solution in IMDM (Life Technologies, Paisley, U.K.), followed by density sedimentation on a cold discontinuous BSA (Path-o-cyte 4, bovine albumin, Instruchemie, Hilversum, The Netherlands) gradient in HBSS (Life Technologies), consisting of layers of 1.5, 2.5, and 5%. The cells at the 2.5–5% interface were harvested and washed in IMDM. These FDC-enriched fractions were used in the experiments.
Cell cultures
All standard media used (IMDM, HBSS) contained gentamicin (90 µg/ml). B cells were cultured for 4 h in 24-well culture plates (Costar, Cambridge, MA) in IMDM supplemented with 10% FCS (HyClone, Logan, UT) in the presence or absence of the general cathepsin inhibitor E64d, the cathepsin B inhibitor CA074-Me (Scientific Marketing Associates, Barnet, U.K.), the cathepsin L inhibitor Z-phenyl-phenyl-CHN2 (ZPP) (Enzyme System Products, Dublin, CA), the cathepsin S inhibitor morpholine urea leucine-homophenylalanine-vinyl sulfone methyl (LHVS) (a kind gift of Dr. Rebecca A.R. Bryant, Harvard Medical School, Boston, MA), and the broad range caspase inhibitor z-Val-Ala-DL-Asp (ZVAD)-fluoromethylketone (Alexis Corporation, Läufelfingen, Switzerland). The inhibitors were dissolved in DMSO (Sigma) and applied at concentrations ranging from 50 nM to 150 µM as indicated.
FDC-enriched cell suspensions were depleted of T cells using SRBCs and cultured in IMDM supplemented with 10% FCS. After 14 h, FDC-B cell clusters were separated from single B cells by 1 x g sedimentation on IMDM with 30% FCS for 30 min at 0°C. Clusters were isolated from the pellet, and single B cells were harvested from the interface. Apoptotic parameters were determined as described below.
Detection of apoptotic parameters
The reduction of mitochondrial membrane potential
(
m) was analyzed according to the method of
Zamzami et al. (26) using 3,3'-dihexyloxacarbocyanine
iodide (DiOC6 (3)) (Molecular Probes, Leiden, The
Netherlands) in combination with propidium iodide (PI) (Sigma). DiOC6
(3) was 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 concentration) was added, and samples were directly
analyzed.
DNA strand breaks were analyzed using the In Situ Cell Death Detection Kit (Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, 1–2 x 106 B cells were washed in PBS with 1% BSA, taken up in PBS and paraformaldehyde (2% final concentration), and permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate. dUTP-fluorescein was coupled to the DNA strand breaks using terminal transferase.
Phosphatidyl serine (PS) exposure was determined using annexin V-FITC (Bender Medsystems Diagnostics, Vienna, Austria) in combination with PI. Cells were labeled with annexin V-FITC for 30 min on ice, washed, and taken up in medium containing PI.
Caspase-3 activity was measured using the ApoAlert CPP32 Fluorescent Assay Kit (Clontech, Palo Alto, CA). Briefly, before or after the induction of apoptosis, cells were lysed and taken up in reaction buffer. After addition of the 7-amino-4-trifluoromethyl coumarin-peptide substrate conjugate and subsequent incubation at 37°C, the enzyme activity was monitored in a JASCO FP-750 spectrofluorometer (B&L Systems, Maarssen, The Netherlands).
Poly(ADP-ribose)-polymerase (PARP) fragmentation was assessed by Western blotting using a rabbit polyclonal Ab (Boehringer Mannheim). Blots were stained with HRP-conjugated goat anti-rabbit IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands) using enhanced chemiluminescence Western blotting detection reagents (Amersham International, Little Chalfont, U.K.).
Endonuclease activity was determined as described by Lindhout et al. (7). Briefly, isolated nuclei were incubated at 37°C for 4 h in TSN buffer (10 mM Tris-HCl, 200 mM sucrose, and 60 mM NaCl (pH 7.5)) followed by the separation of large and small DNA fragments; these fragments were precipitated and run on an agarose gel.
Fluorescence analyses of DiOC6 (3) signal, DNA strand breaks, and annexin V-FITC staining were conducted on a FACScan (Becton Dickinson, Mountain View, CA) and analyzed using WinMDi 2.7 software developed by Joseph Trotter (Scripps Research Institute, La Jolla, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Apoptotic parameters were measured and compared in F-B, in B cells
clustered to FDCs (C-B), and in nonclustered B cells (N-B) that were
separated from each other by 1 x g sedimentation
on IMDM with 30% FCS. Several apoptotic parameters were analyzed: PS
exposure using annexin V-FITC staining (Fig. 1
a), reduction of
m using DiOC6 (3) staining
(Fig. 1b), DNA strand breaks using the in situ cell death
detection method (Fig. 1c), caspase-3 activity by
monitoring the cleavage product of the caspase-3 specific substrate
7-amino-4-trifluoromethyl coumarin-peptide substrate conjugate
(Fig. 2a), cleavage of PARP
(Fig. 2b), and endonuclease activity in isolated nuclei of
GC B cells (Fig. 2c).
|
|
, a–c, F-B showed low levels of PS
exposure, a high m, and very few DNA strand
breaks. Caspase-3 is inactive in these cells (Fig. 2a),
which is consistent with the finding that the DNA repair enzyme PARP is
present in its uncleaved 113-kDa form (Fig. 2b). These data
confirm the nonapoptotic phenotype of F-B.
After 14 h however, the single B cell fraction (N-B) showed a
largely increased level of PS exposure, reduced
m, and increased numbers of DNA strand
breaks (Fig. 1, a–c). Moreover, caspase-3 activity could be
demonstrated at this timepoint (Fig. 2a). This is in
agreement with the finding that PARP was now present as an inactivated
89-kDa fragment (Fig. 2b).
By contrast, the C-B showed levels of PS exposure,
m, DNA strand breaks, PARP cleavage, and
caspase-3 activity that were similar to the F-B.
Interestingly, endonuclease activity could be demonstrated in the
nuclei of both F-B (Fig. 2c, lane 2) and the
single B cell fraction (N-B) (Fig. 2c, lane 3).
In contrast, endonuclease activity (Fig. 2c, lane
4) could not be demonstrated anymore in isolated nuclei of
C-B.
These data clearly show that FDCs keep the entire apoptotic machinery in GC B cells silent and furthermore switch off the latent endonuclease activity in the nuclei of the GC B lymphocytes.
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
absence of various cathepsin inhibitors and the general caspase
inhibitor ZVAD (Fig. 3). DNA strand
breaks were analyzed after 4 h using the in situ cell death
detection assay. Strikingly, DNA strand breaks were blocked not only
with the caspase inhibitor ZVAD but also with the general cathepsin
inhibitor E64d. However, this was not observed when specific inhibitors
for cathepsin B (CA074-Me), cathepsin L (ZPP), or cathepsin S (LHVS)
were used. These data imply that both caspase and cathepsin activities
are required for the occurrence of DNA strand breaks in GC B cells, and
that caspase(s) and cathepsin(s) must act in a sequential manner.
|
To further investigate at which level the cathepsins were involved
in the apoptotic cascade, the effects of the cathepsin inhibitor E64d
and the caspase inhibitor ZVAD on different apoptotic processes were
measured, including PS exposure, reduction of
m, caspase-3 activity, cleavage of PARP,
and DNA strand breaks. As shown in Fig. 4a, PS exposure was inhibited
by ZVAD but not by E64d. Also, ZVAD inhibited mitochondrial damage
(Fig. 4b), caspase-3 activity (Fig. 4c), and
processing of the caspase-3 substrate PARP (Fig. 4d). E64d
did not block any of these processes, indicating that cathepsin
activity could not reside upstream of caspase activity. Clear
inhibition by E64d was only found when DNA strand breaks were studied
(Fig. 4e). These data demonstrate that a thus far
unidentified cathepsin activity is involved in one of the very
downstream steps of the apoptotic machinery, and that this activity is
essential for the execution of DNA fragmentation. Consequently,
cathepsin activity must be downstream of caspase-3 activity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Thus far, the enzymatic activities that are operational during GC B cell apoptosis as well as the mechanisms used by FDCs to rescue the binding B lymphocytes are poorly understood. Many factors contribute to the rescue process, but none of these, either alone or in concert, seem to explain the rapid switch-off of nuclear endonuclease in GC B cells by FDCs. Obviously, BCR cross-linkage with Ag present in immune complexes on the surface of FDCs must be implicated as a first step in the binding process in vivo. Subsequent interactions of adhesion molecules, including LFA-1/ICAM-1 and VLA-4/VCAM-1, will establish firm contact between FDC and GC B cells. The relevance of these interactions for B cell survival has been shown in many studies in which B cell binding was impaired and/or apoptosis was delayed or inhibited by Abs to the BCR, LFA-1, VLA-4, ICAM-1, or VCAM-1 (5, 8, 9, 10). However, the cross-linkage of these molecules, alone or in combination, could not entirely switch off the apoptotic capacity of the B cell (i.e., it failed to block the latent endonuclease in their nuclei) (7). At this stage, the rescue process is essentially CD40 independent; although CD40 ligation postpones GC B cell death, it cannot eliminate the endonuclease activity (7). Consequently, some additional, unique signal given by FDCs must be implicated for the definitive switch-off of this endonuclease.
The present paper confirms and extends previous studies demonstrating this unique function of FDCs (7). In the absence of FDCs in vitro, GC B lymphocytes show several different aspects of apoptosis, including PS exposure, mitochondrial inactivation, caspase-3 activity, PARP cleavage, and DNA fragmentation. All of these processes are cancelled upon binding to FDCs. Remarkably, FDCs prevent the up-regulation of multiple, caspase-related events. In addition, they eliminate the latent endonuclease activity that is present in the nuclei of freshly isolated B lymphocytes.
We found that caspase-3 activity is absent in both freshly isolated and FDC-bound GC B cells. Concordantly, PARP is found in its intact form only. These data are in agreement with our previous experiments showing that GC B lymphocytes are resistant to Fas-mediated apoptosis as long as they remain in contact with FDCs (27). The reason for this resistance is still unclear, but it may be speculated that inhibitory proteins such as FLICE-inhibitory protein or Fas apoptosis inhibitory molecule (28, 29) are instrumental in keeping the caspase route silent. Detachment of GC B cells from their natural counterstructures may lead to a reactivation of the caspase route, resulting in the execution of apoptosis. Recently, it was reported that a caspase-activated DNase/DNA fragmentation factor (DFF)40 is released from its inhibitor, inhibitor of caspase-activated DNase/DFF45, by cleavage through caspase-3 (17, 18, 19). These findings connect the caspase route directly to the execution of apoptosis (i.e., to DNA fragmentation). Thus far, our attempts to demonstrate DFF45 processing in GC B cells by Western blotting have been unsuccessful (data not shown). This may mean either that a different DNase activity is used by these cells or that the activation mechanism of the DNase is different.
Here, we have shown that an additional protease (i.e., a thus far unidentified cathepsin) is part of the apoptotic cascade of GC B lymphocytes. Inhibition of this cathepsin with the inhibitor E64d completely prevents DNA strand breaks but leaves the caspase-dependent PS exposition and PARP cleavage untouched, demonstrating that the cathepsin acts downstream of caspase-3.
It is tempting to speculate that blockade of this very downstream cathepsin activity may well be the target of FDC action. Natural inhibitors of cathepsins belong to the family of cystatins (30, 31), and it has been demonstrated that cathepsins and cystatins are involved in different models of apoptosis. For instance, in a model for bile-salt induced apoptosis in hepatocytes, it was shown that cathepsin B was downstream of caspase-3. Cystatin A could efficiently block apoptosis in this model (32). In addition, increased apoptosis was reported in the brain of the cystatin B knockout mouse (33). Moreover, it was shown that cystatin A could inhibit the virus-induced apoptosis of a carp cell line (34).
Interestingly, cystatin A is present in FDCs (35); it is attractive to consider the possibility that it is transported to B cells during the intimate contact with FDCs. Currently, we are investigating this protein and its relevance in FDC-mediated antiapoptotic signaling in GC B lymphocytes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: GC, germinal center; FDC, follicular dendritic cell; F-B, freshly isolated B cells; N-B, nonclustered B cells; C-B, B cells clustered to FDCs; BCR, B cell receptor; ZPP, Z-phenyl-phenyl-CHN2; LHVS, leucine-homophenylalanine-vinyl sulfone methyl; ZVAD, z-Val-Ala-DL-Asp; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; PI, propidium iodide; PS, phosphatidyl serine; PARP, poly(ADP-ribose)-polymerase; LFA, lymphocyte function-associated Ag; VLA, very late Ag; m, mitochondrial membrane potential; DFF, DNA fragmentation factor.
Received for publication April 13, 1999. Accepted for publication June 11, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. A. G. M. Cillessen, J. C. Reed, K. Welsh, C. Pinilla, R. Houghten, E. Hooijberg, J. Deurhof, K. C. M. Castricum, P. Kortman, C. J. Hess, et al. Small-molecule XIAP antagonist restores caspase-9 mediated apoptosis in XIAP-positive diffuse large B-cell lymphoma cells Blood, January 1, 2008; 111(1): 369 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A.G.M. Cillessen, C. J. Hess, E. Hooijberg, K. C.M. Castricum, P. Kortman, F. Denkers, W. Vos, M. A. van de Wiel, G. J. Schuurhuis, G. J. Ossenkoppele, et al. Inhibition of the Intrinsic Apoptosis Pathway Downstream of Caspase-9 Activation Causes Chemotherapy Resistance in Diffuse Large B-Cell Lymphoma Clin. Cancer Res., December 1, 2007; 13(23): 7012 - 7021. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. van Nierop, F. J.M. Muller, J. Stap, C. J.F. Van Noorden, M. van Eijk, and C. de Groot Lysosomal Destabilization Contributes to Apoptosis of Germinal Center B-lymphocytes J. Histochem. Cytochem., December 1, 2006; 54(12): 1425 - 1435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Huber, C. Thielen, H. Seeger, P. Schwarz, F. Montrasio, M. R. Wilson, E. Heinen, Y.-X. Fu, G. Miele, and A. Aguzzi Lymphotoxin-{beta} Receptor-Dependent Genes in Lymph Node and Follicular Dendritic Cell Transcriptomes J. Immunol., May 1, 2005; 174(9): 5526 - 5536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Meyer-Hermann and P. K. Maini Cutting Edge: Back to "One-Way" Germinal Centers J. Immunol., March 1, 2005; 174(5): 2489 - 2493. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Jackson and J. D. Capra IgH V-Region Sequence Does Not Predict the Survival Fate of Human Germinal Center B Cells J. Immunol., March 1, 2005; 174(5): 2805 - 2813. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-S. Park, S.-O. Yoon, R. J. Armitage, and Y. S. Choi Follicular Dendritic Cells Produce IL-15 That Enhances Germinal Center B Cell Proliferation in Membrane-Bound Form J. Immunol., December 1, 2004; 173(11): 6676 - 6683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-C. Michallet, F. Saltel, M. Flacher, J.-P. Revillard, and L. Genestier Cathepsin-Dependent Apoptosis Triggered by Supraoptimal Activation of T Lymphocytes: A Possible Mechanism of High Dose Tolerance J. Immunol., May 1, 2004; 172(9): 5405 - 5414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-C. Michallet, F. Saltel, X. Preville, M. Flacher, J.-P. Revillard, and L. Genestier Cathepsin-B-dependent apoptosis triggered by antithymocyte globulins: a novel mechanism of T-cell depletion Blood, November 15, 2003; 102(10): 3719 - 3726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-L. Marchand, S. Der Sarkissian, P. Hamet, and D. deBlois Caspase-Dependent Cell Death Mediates the Early Phase of Aortic Hypertrophy Regression in Losartan-Treated Spontaneously Hypertensive Rats Circ. Res., April 18, 2003; 92(7): 777 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Thorburn, L. M. Bender, M. J. Morgan, and A. Thorburn Caspase- and Serine Protease-dependent Apoptosis by the Death Domain of FADD in Normal Epithelial Cells Mol. Biol. Cell, January 1, 2003; 14(1): 67 - 77. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Beyer, M. Meyer-Hermann, and G. Soff A possible role of chemotaxis in germinal center formation Int. Immunol., December 1, 2002; 14(12): 1369 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Matarrese, L. Di Biase, L. Santodonato, E. Straface, M. Mecchia, B. Ascione, G. Parmiani, F. Belardelli, M. Ferrantini, and W. Malorni Type I Interferon Gene Transfer Sensitizes Melanoma Cells to Apoptosis via a Target Activity on Mitochondrial Function Am. J. Pathol., April 1, 2002; 160(4): 1507 - 1520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. van Eijk, J. P. Medema, and C. de Groot Cutting Edge: Cellular Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Inhibitory Protein Protects Germinal Center B Cells from Apoptosis During Germinal Center Reactions J. Immunol., June 1, 2001; 166(11): 6473 - 6476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hennino, M. Berard, P. H. Krammer, and T. Defrance FLICE-inhibitory Protein Is a Key Regulator of Germinal Center B Cell Apoptosis J. Exp. Med., February 12, 2001; 193(4): 447 - 458. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Katz, M. R. Deehan, S. Seatter, C. Lord, R. D. Sturrock, and M. M. Harnett B Cell Receptor-Stimulated Mitochondrial Phospholipase A2 Activation and Resultant Disruption of Mitochondrial Membrane Potential Correlate with the Induction of Apoptosis in WEHI-231 B Cells J. Immunol., January 1, 2001; 166(1): 137 - 147. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
