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*
Temple University School of Medicine, Philadelphia, PA 19140;
Johns Hopkins University School of Medicine, Baltimore, MD 21218;
Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637; and
Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The critical need for CD95 aggregation to initiate signaling for apoptosis is evidenced by several studies. Soluble CD95 ligand that can only trimerize CD95 (5, 6) fails to process caspase-8 (7). However, cross-linking of receptor or pro-caspase fusion proteins induces death (8, 9), demonstrating that multimerizing CD95is necessary to potentiate the inherent proteolytic activity of caspase-8 to transactivate a closely neighboring caspase. Earlier results comparing the aggregating IgG3 CD95 agonistic anti-APO-1 Ab with isotype-switched nonaggregating Abs also allude to the possibility that receptor oligomerization is necessary to activate caspase-8 (10). Based on this, we asked whether PI 3'-K mediated its anti-apoptotic effects on T cells by preventing CD95 aggregation. Indeed, we show here that PI 3'-K inhibits caspase-8 cleavage by inhibiting aggregation of CD95. This inhibition is effected by an actin-dependent reduction in the lateral diffusion of CD95.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tetanus toxoid-reactive Th2 clones and purified protein
derivative-reactive Th1 clones were established by limiting dilution
technique as previously described (11). Subset specificity
of the clones was determined by measuring production of IFN-
and
IL-4. For each T cell clone, 1 x 105 cells
were added in 1 ml of Iscove’s modified DMEM and 10% human AB
serum in 24-well tissue culture plates precoated with 2.5 µg of Ab to
CD3 (BioSource International, Camarillo, CA). Supernatants from the
stimulated cells were harvested 18–20 h later, and cytokine quantities
were measured by sandwich enzyme immunoassay using appropriate pairs of
capture Abs and biotinylated detecting Abs (BD Pharmingen, San Diego,
CA). To expand cell numbers, clones were maintained in IL-2,
with bi-weekly stimulation of Ag and autologous APCs. Cytokine profile
of the clones was routinely reconfirmed after several rounds of
stimulation. We have observed that even after several in vitro
stimulations and expansions, the cytokine profile and Ag reactivity of
the clones remain unchanged.
Abs and reagents
The anti-APO-1 monoclonal (IgG3, 
) is an
agonistic Ab recognizing an extracellular epitope of CD95
(12). The anti-caspase-8 Abs used recognize two
different functional domains of caspase-8. The N2 Ab recognizes an
epitope adjoining the tandem death effector domain containing
prodomain. The C15 Ab detects the catalytically active p18 large
subunit (13). Biotinylated and FITC-conjugated DX11
anti-CD95 mAb was obtained from BD Pharmingen. Caspase-8 encoding
plasmid was a gift from Vishwa Dixit (Genentech, San Francisco,
CA). In vitro transcription and translation was performed using
the TnT Quick Coupled System obtained from Promega (Madison, WI).
Wortmannin and cytochalasin D were purchased from Sigma (St. Louis, MO)
and used at a final concentration of 1 µM.
DISC analysis and Western blotting
Association and cleavage status of caspase-8 at the DISC was determined in 107 T cells obtained after varying treatment conditions. Following the varying treatment conditions, T cells were harvested, washed in cold buffer, pelleted, and lysed in ice-cold lysis buffer (as described in Refs. 2, 3) for 30 min. Cell debris was pelleted by centrifugation for 10 min at 13,000 x g. Lysates were supplemented with 50 µl of rabbit anti-mouse (Cappel, Cochranville, PA)-coated staphylococcus A (Sigma). DISCs were allowed to immunoprecipitate in the cold for at least 1 h, then were washed in cold TNE (1 M Tris (pH 8), 1.5 M NaCl, 0.5 M EDTA, 1% Nonidet P-40) buffer. Immune complexes and staphylococcus A were separated after six washes by 15 min incubation in 2-ME containing sample buffer, staphylococcus A was spun out, and supernatants containing the immunoprecipitates were boiled. For Western blotting, lysates were separated on 12% SDS-PAGE, transferred to supported nitrocellulose membrane (Bio-Rad, Hercules, CA), and blocked with nonfat milk-containing Tween 20 + TBS (TTBS). Blots were incubated with primary Ab diluted in TTBS overnight at 4°C, washed three to four times for 10 min each with TTBS, then detected with HRP-conjugated secondary Ab, and developed using the enhanced chemiluminescence method (Amersham, Buckinghamshire, U.K.) following the manufacturer’s protocol. Multimeric status of CD95 was determined by fractionating either immunoprecipitated DISC or whole cell lysates under nonreducing conditions on a 5–15% gradient gel (Jule, New Haven, CT), Western blotted using the biotinylated DX11 mAb, and visualized with streptavidin-coupled HRP and ECL substrate.
In vitro caspase-8 cleavage assay
In vitro transcribed/translated 35S-methionine-labeled caspase-8 was incubated with immunoprecipitated DISC obtained from 30 x 106 T cell clones resuspended in 50 µl cleavage assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10 mM DTT, 20% sucrose) (13) for 24 h at 4°C. Following incubation, pellets and supernatants were separated by centrifugation. Pellets were washed five times in DISC lysis buffer, resuspended in reducing sample buffer, and applied to a 12% SDS-PAGE gel. Supernatants were similarly subjected to SDS-PAGE separation. Gels were dried and exposed to film. Alternatively, gels were transferred to nitrocellulose and probed with caspase-8-specific Abs. Quantitative analysis of in vitro cleavage was determined by incubating cleavage assay supernatant with the caspase-8 colorimetric substrate Ile-Glu-Thr-Asp-p-nitroaniline (IETD-pNA) (BioVision, Palo Alto, CA) per manufacturer instructions.
Fluorescence recovery after photobleaching (FRAP)
Lateral diffusion was measured using the technique of FRAP (14). CD95 on T cell clones was fluorescently labeled with the nonagonistic DX11 Ab. Cells were washed twice before being loaded into 0.05 mm path length glass capillaries (Vitro Dynamics, Rockaway, NJ) and mounted onto glass slides with nail polish. Measurements of lateral diffusion and mobility were taken on a Zeiss Axioplan fluorescence microscope using a 1.30 63X NA Zeiss Plan Neo-fluor objective to focus a 488 nm argon ion laser. Data were collected and analyzed using custom software (14). Diffusion coefficient is expressed as 10-10 cm2/s.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first compared DISCs immunoprecipitated from extracts of Th2
cells on the path to apoptosis after cross-linking of CD95 with DISCs
from Th2 cells that were protected from apoptosis by concomitant
ligation of CD3. Immunoprecipitated DISCs were separated under
nonreducing conditions on a gradient gel to visualize receptor
aggregation (Fig. 1
A).
SDS-stable high molecular mass tetrameric aggregates of CD95 were
isolated from cells whose CD95 was ligated with anti-APO-1 Abs
(Fig. 1A, lane 1). In contrast, cells treated with
anti-APO-1 and anti-CD3 did not yield SDS- stable CD95
aggregates (Fig. 1A, lane 2), but only receptor dimers. When
PI 3'-K activity was inhibited with wortmannin, tetrameric CD95
aggregates were isolated from cells treated with both anti-CD3 and
anti-APO-1, indicating that conditions that induce apoptosis lead
to formation of higher order receptor oligomers (Fig. 1A, lane
3). The CD95 aggregates were 
200 kDa in size, suggesting that
formation of an active complex required a minimum of four CD95
molecules to aggregate in the membrane. This aggregation is ligand
induced because only monomeric CD95 was isolated from control
unstimulated cells (data not shown). Preassociated trimers
(15) were not observed, as no chemical cross-linking was
performed to preserve the trimeric structures in the absence of
ligand.
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B, lane 1). In contrast, DISCs isolated from
anti-CD3- and anti-APO-1-stimulated cells only processed
exogenous caspase-8 to the p41/43 fragment, and failed to produce the
p26 subunit (Fig. 1B, lane 2). Cells treated with wortmannin
before ligation of CD3 and CD95 were not restricted in their ability to
process exogenous caspase-8, and generated the p26 prodomain fragment
(Fig. 1B, lane 3). As reported previously, we failed to
immunoprecipitate DISCs from cells that received only CD3 stimulation
(data not shown). Complete cleavage of exogenous caspase-8 was
confirmed by examining the supernatants of the in vitro cleavage assay
for the presence of catalytically active p18 subunit. Only under
conditions where p26 fragment was generated was the presence of p18
also observed (Fig. 1C, lanes 1 and 3).
Conditions that lead to an active DISC also led to a 6-fold increase in
caspase-8 activity measured by cleavage of IETD-pNA, whereas conditions
where cells cannot generate active DISC and are protected from death
failed to cleave IETD-pNA (Fig. 1D). Thus DISCs from cells
that are susceptible to CD95 triggered death, and can fully process
caspase-8, consist of SDS-stable aggregates of CD95, whereas DISCS from
cells protected from apoptosis by activation of PI 3'-K lack the CD95
aggregates necessary for full DISC activity and complete caspase-8
cleavage. T cell activation alters CD95 lateral diffusion in the plasma membrane by a PI 3'-K-dependent mechanism
The lack of CD95 aggregates in CD3-stimulated cells raised the
possibility that PI 3'-K inhibits lateral diffusion of CD95 and so
reduces the probability of their aggregation and formation of
functional DISCs. Therefore, we compared the lateral diffusion of CD95
in untreated and CD3-stimulated cells using FRAP. CD95 receptors of
three individual T cell clones were fluorescently labeled with the
nonagonistic CD95 Ab, DX11. A mobile fraction (40–50% of the total)
of CD95 molecules diffused in the membrane of unstimulated cells with a
diffusion coefficient, D30 x 10-10
cm2/s (Fig. 2, left panel). In contrast, after CD3 ligation, D of CD95 from
a parallel set of T cell clones was 1 order of magnitude smaller,
3 x 10-10 cm2/s
(Fig. 2, middle panel); the mobile fraction of receptors
remained unchanged (data not shown). The decrease in D for CD95
following CD3 ligation, which correlated with the inability of
CD3-stimulated cells to rapidly form CD95 aggregates, suggested that PI
3'-K plays a role in regulating the lateral diffusion of CD95 movement
in the plasma membrane. Consistent with this idea, inhibition of
CD3-triggered PI 3'-K activity by wortmannin returned D to values
observed for unstimulated, apoptosis-sensitive cells (Fig. 2, right panel).
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60 x
10-10 cm2/s (Fig. 2B), comparable with unstimulated Th2 clones. However, in
contrast to what we had observed in Th2 clones, CD3 stimulation did not
alter CD95 lateral diffusion in Th1 cells (Fig. 2B). Actin cytoskeleton regulates CD95 aggregate formation and susceptibility to CD95-signaled death
The role of PI 3'-K in regulating cytoskeletal networks is well documented (16, 17, 18, 19, 20, 21). The 10-fold reduction in D after CD3-stimulated PI 3'-K activation is consistent with steric hindrance of receptor movement by the cortical actin cytoskeleton (22, 23). Because the defect in caspase-8 processing correlates with hindered diffusion of CD95 to move in the membrane, we investigated the possibility that the cytoskeleton may be responsible for regulating CD95 movement.
To establish the linkage between PI 3'-K regulated CD95 aggregation and
the cytoskeleton, we determined whether apoptosis, CD95 aggregation,
caspase-8 cleavage, and CD95 lateral diffusion were all affected by
cytochalasin D, which disrupts actin filaments. Cells that were treated
with cytochalasin D, before CD95 ligation, were no longer protected
from apoptotic death by CD3 ligation (Fig. 3). Lysates of these cells also yielded
SDS-stable aggregates of CD95 (Fig. 4A, lane 3), like untreated
CD95-ligated cells (Fig. 4A, lane 1). In contrast, cells
that were CD3-stimulated before anti-APO-1 treatment, but not
treated with cytochalasin D and were protected from apoptosis, did not
yield CD95 aggregates (Fig. 4A, lane 2). DISCs from
CD3-activated T cells, treated with cytochalasin D just before
CD95 ligation, fully processed caspase-8 (Fig. 4B, lane 3)
just as well as unactivated CD95-ligated cells (Fig. 4B, lane
1) as evidenced by the presence of the p26 prodomain at the DISC,
whereas CD3 activation without cytochalasin D treatment before CD95
ligation led to incomplete cleavage and an absence of p26 prodomain
fragment at the DISC (Fig. 4B, lane 2). Cytochalasin D
treatment of activated cells also returned the diffusion coefficient of
CD95 to that seen in untreated cells (Fig. 5) or of wortmannin-treated
CD3-stimulated cells, 30 x 10-10
cm2/s-1 (Fig. 2).
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). However, cytochalasin treatment
following CD3 ligation did not abrogate PI 3'-K activity in the cells
(Fig. 6). At present we are not sure regarding the apparent synergy in
PI 3'-K activity when CD3 and cytochalasin are used. Following CD3
activation, signaling molecules accumulate in rafts, and it is possible
that the addition of cytochalasin excludes inhibitory molecules such as
Cbl-b (24). However, of significance to this study is the
fact that despite the presence of active PI 3'-K, when cytoskeletal
constraints are removed CD95 receptors can diffuse freely to form
tetrameric aggregates that are capable of fully processing caspase-8
and sensitizing cells to apoptosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although slow receptor diffusion could, with time, result in tetrameric aggregate formation, a competing process such as receptor internalization (28) may remove nascent receptor aggregates before they reach the critical size. Alternatively, inactivation or dispersion of nascent DISCs could occur. In this regard, FLIP (cellular FLICE inhibitory protein), a catalytically inactive form of caspase-8, which is also competitively recruited to the DISC (1, 2), can inactivate the nonmultimerized DISC by initiating an anti-apoptotic signaling response from CD95 (29). In addition, a short splice variant of FLIP is up-regulated in activated T cells and is strongly recruited to the DISC, where it blocks caspase-8 cleavage (30). The exact stoichiometry of caspase-8 and FLIP molecules that get recruited to the DISC following CD95 receptor ligation is undetermined. Nevertheless, the fact that both FLIP and caspase-8 molecules are capable of getting recruited into the DISC necessitates rapid CD95 aggregate formation to allow adequate caspase-8 molecules to be present in the DISC for efficient trans-proteolytic activation. Hence the ability to rapidly form aggregates is critical in determining the outcome of CD95 ligation.
Diffusion coefficient of many membrane proteins appears to depend on their exodomains (31, 32, 33). Therefore, regulation of D, rather than mobile fraction by the cytoskeleton is unusual, and contrasts with the results on aggregation of Fc receptors (34, 35, 36) and CD2 (37), in which aggregated receptors interact with the cytoskeleton and are immobilized and internalized. Because we do not see changes in the mobile fraction of CD95 following CD3 ligation, it is unlikely that lateral diffusion of CD95 is impaired by direct tethering to the cytoskeleton. We instead propose that lateral diffusion of CD95 in the membrane is physically impeded due to corralling by reorganized actin cytoskeletal networks. Studies of the interactions between the erythrocyte membrane protein Band 3 and the erythrocyte membrane cytoskeleton support a paradigm very similar to the one we propose for CD95 (22, 23). In this model, it is suggested that the cytoplasmic portion of Band 3 collides with the mesh of the membrane cytoskeleton and becomes fenced within the actin cytoskeletal grid, resulting in slowed diffusion in the membrane (23). In a like manner, lateral diffusion of transferrin receptor molecules in the plasma membrane of cells are also regulated by the dynamically fluctuating membrane cytoskeleton (38).
A recent study by Siegel et al. (15) revealed that trimerization of CD95 is not ligand dependent, but preassociated trimers exist on the cell surface of T cells. Sixty percent of the CD95 chains exist in the preassociated trimeric state, but are unable to recruit caspase-8 and initiate signaling for apoptosis. Our results show that CD95 has to minimally tetramerize to recruit and cleave caspase-8 to its catalytically active form. Taken together, these data imply that preassociated trimers have to encounter another monomeric receptor to form active complexes in response to treatment with agonistic APO-1 Abs. Following CD3-stimulation, diffusion of both monomers and trimers is restricted, preventing the formation of active tetramers. At present it is not clear how dimers are formed in CD3-stimulated cells following APO-1 treatment. A possible explanation is that, despite the caging effect induced by the polymerized actin in CD3-stimulated cells, there is likely a power law relationship between frequency of correctly sized gaps in the cytoskeleton and size of the diffusing molecules that allows monomers to diffuse slightly faster than trimers.
In conclusion, the findings reported here describe a novel mechanism for how PI 3'-K regulates CD95 function by a dynamic membrane process. Unlike most receptors that can signal following either their dimerization or trimerization, we show that CD95 has to become aggregated into tetramers to initiate the apoptotic signaling pathway. The necessity for receptor aggregation favors modulation of lateral diffusion as a unique mechanism to regulate CD95 function.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Padmini Salgame, Temple University School of Medicine, Department of Microbiology and Immunology, Kresge, Room 501, 3400 North Broad Street, Philadelphia, PA 19140. E-mail address:salgame{at}astro.temple.edu
3 Abbreviations used in this paper: DISC, death-inducing signaling complex; PI 3'-K, phosphatidylinositol 3'-kinase; FRAP, fluorescence recovery after photobleaching; TTBS, Tween 20 + TBS; IETD-pNA, Ile-Glu-Thr-Asp-p-nitroaniline; FLIP, cellular FLICE inhibitory protein.
Received for publication December 8, 2000. Accepted for publication March 21, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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