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Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105
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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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RI (7), CD95
(8, 9, 10), CD28 (11), TNFR (12),
platelet-derived growth factor receptor (13),
CD2, CD44, L-selectin, or integrins (14). Many receptor
molecules cluster in distinct cholesterol- and sphingolipid-rich
domains of the cell membrane termed rafts (15, 16, 17). The
self-association of sphingolipids and cholesterol favors the
formation of a liquid-ordered status that separates sphingolipids and
cholesterol from phospholipids in the cell membrane and finally forms
raft domains (17). Those domains were also named
detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs),
because they are insoluble in some nonionic detergents. Some receptor
molecules are constitutively present in those membrane rafts, whereas
other receptor molecules translocate into rafts upon stimulation
(16, 18); for example, the BCR (4, 19) or the
FcRI (7) reside in the detergent-soluble membrane
fraction before cognate stimulation and translocate into
detergent-insoluble rafts within minutes after ligation. Translocation
and clustering of the BCR or the FcRI in those rafts appears to be
required for activation of early signaling events initiated by the BCR
(4) or the FcRI (7). In addition to the
ability to recruit receptor molecules, sphingolipid-rich rafts also
contain many signaling molecules, including Src-like tyrosine kinases
(20) or phospholipases (21). Finally, raft
structures appear to exclude other (inhibitory) enzymes, e.g., the
tyrosine phosphatase CD45 (4, 22). The selective
interaction of clustered receptor molecules with signaling molecules in
rafts probably constitutes a central element in the initiation of
receptor signaling. We have recently suggested a new concept for receptor clustering using CD95 as the paradigm (9, 10, 23). CD95 stimulation triggers a translocation of acid sphingomyelinase (ASM) from intracellular stores onto the extracellular leaflet of the cell membrane (9). Electron microscopy studies localized the ASM in intracellular vesicles of unstimulated cells (23) and exposure of the ASM on the extracellular leaflet of the cell membrane is most likely mediated by the fusion of those vesicles (9, 23) with the cell membrane upon CD95 stimulation. Surface ASM localized to sphingolipid-rich rafts released extracellularly oriented ceramide and triggered a clustering of CD95 upon stimulation of lymphocytes via CD95 (9, 10). Ceramide was required and sufficient to trigger the clustering of CD95 in sphingolipid-rich signaling platforms. Previous studies (24, 25) on IL-1 and p75 nerve growth factor receptors support the notion of ASM activity in rafts. The exact identity of surface ASM is still unknown, because cells contain at least two isoforms of ASM, a lysosomal ASM and a secretory ASM (26, 27). The two forms are derived from the same gene, but are differentially processed at the amino terminus and display a different glycosylation pattern (27). Whether surface ASM represents a third isoform or a specialized form of the secretory or lysosomal ASM, respectively, has to be determined.
CD95 exhibits sequence homology to CD40 (28) raising the
question of whether ASM and ceramide are involved in signaling and
clustering of CD40. CD40 has been shown to be central in the
stimulation of B cells, because B lymphocytes lacking signals via CD40
are unable to switch from IgM to IgG synthesis upon antigenic
stimulation (29, 30). Ligation of CD40 results in the
association of the receptor with several intracellular proteins
including TNFR-associated factors (TRAFs; Refs. 31 and
32 ; for review see Ref. 33) and Janus family
kinase 3 (JAK3; Ref. 34). TRAFs couple CD40 to an
activation of NF-
B (35) and extracellular-regulated
kinase (36), JAK3 to the STATs (34), and
thus, finally to gene transcription. In addition, CD40 stimulation
triggers an activation of several Src-like tyrosine kinases, in
particular Lyn (37), Blk (38), and Fyn
(39), as well as of the protein tyrosine kinase, Syk
(39). Activation of these kinases by CD40 correlates with
a release of inositol-3-phosphate (37), stimulation of Ras
(38), extracellular-regulated kinases (38, 40), c-Jun N-terminal kinases (JNKs; Refs. 40, 41, 42),
and p38 mitogen-activated protein kinase (42). However,
most, if not all, of these signaling events seem to depend on the
clustering and recruitment of CD40 in sphingolipid-rich rafts, at least
in dendritic cells (5) and B lymphocytes (6).
Therefore, we aimed in the present study to define mechanisms mediating
the aggregation of CD40 in rafts and the formation of a larger CD40
cluster. To this end, we investigated the role of the ASM and ceramide
released in sphingolipid-rich membrane rafts for CD40 clustering.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human JY B lymphocytes, Niemann-Pick disease type A (NPDA) B lymphocytes, or Farber B lymphocytes were transfected with expression plasmids encoding ASM, vesicular stomatitis virus (VSV)-tagged ASM, Myc-tagged ASM, acid ceramidase (AC), or a control vector. NPDA cells were obtained from a patient with NPDA disease and Farber B cells were provided by Dr. K. Ferlinz (University of Bonn, Bonn, Germany). To retransfect the cells, we subcloned cDNAs encoding unmodified ASM, VSV-tagged ASM, and AC into the pJK vector. These subclones were designated pJK-asm, pJK-vsv-asm, and pJK-ac, respectively (43, 44). The pJK vector contains an additional gene that encodes a single-chain Ab fused with a Myc-tag to permit isolation of transfected cells by panning with the anti-Myc 9E10 Ab. In addition, the cDNA fragments encoding unmodified ASM and Myc-tagged ASM were subcloned into the pEF vector and designated pEF-asm and pEF-myc-asm, respectively. Both pJK and pEF vectors contain the elongation factor promoter to induce constitutive expression of ASM. Transient transfection of NPDA B lymphocytes was performed by using a BTX electroporator (Genetronics, San Diego, CA) that administered five 99-µs pulses at 550 V. Stably transfected cells were selected with 0.75 mg/ml G418, which was removed at least 24 h before any signaling studies were performed. Expression of ASM constructs was confirmed by flow cytometry and by measurements of ASM activity that indicated an expression level comparable to that in normal control JY B cells.
All cells were grown in RPMI 1640 supplemented with 10% FBS, 10 mM of HEPES (pH 7.4), 2 mM of L-glutamine, 1 mM of sodium pyruvate, 100 µM of nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM of 2-ME (Life Technologies, Karlsruhe, Germany). The mouse IgG1 anti-CD40 mAb (clone 5C3) was obtained from BD PharMingen (San Diego, CA). Anti-VSV P5D4 and anti-Myc 9E10 Abs were obtained from Roche (Mannheim, Germany), the anti-ceramide 15B4 mAb was obtained from Alexis (Grunberg, Germany), choleratoxin and anti-choleratoxin Abs from Sigma-Aldrich (Deisenhofen, Germany), the anti-ASM antiserum was kindly provided from Dr. K. Sandhoff (University of Bonn). Unless otherwise indicated, anti-CD40 5C3 Abs were added to the cells at a concentration of 100 ng/ml for 10 min at 4°C, cells were washed and then stimulated by the addition of 1 µg/ml F(ab')2 fragments of anti-mouse Ig (BD PharMingen) at 37°C. The time points given in the results or the figures, respectively, refer to the time with anti-mouse Ig stimulation.
To disrupt sphingolipid- and cholesterol-rich rafts, we used 10 µg/ml
nystatin, 0.5 µg/ml filipin, or 1 mM of 
-cyclodextrin. These
drugs, which destroy sphingolipid-enriched rafts by disrupting
cholesterol metabolism, were added 15 min before stimulation by
anti-CD40. 
-cyclodextrin (1 mM) was used as control.
C16-ceramide, C20:4
arachidonic acid, sphingomyelin (SM),
dihydro-C2-ceramide, and
dihydro-C16-ceramide were dissolved in 0.01%
octyl-glucopyranoside and used at 1 µM.
Fluorescence microscopy
For confocal and conventional fluorescence microscopy, lymphocytes were immobilized for 15 min on glass coverslips coated with 1% (v/v) poly L-lysine, washed, incubated with 100 ng/ml anti-CD40 5C3 Ab for 10 min at 4°C, washed again and stimulated for the indicated time with 1 µg/ml F(ab')2 anti-mouse Ig as above. Unstimulated cells were incubated with an irrelevant mouse IgG1 instead of anti-CD40 Abs. After the indicated time of stimulation, the cells were washed and fixed for 15 min in 1% (w/v) paraformaldehyde in PBS. Cells were washed and incubated with an irrelevant rabbit or human Ig (20 µg/ml) for 45 min to block FcR before the addition of the primary Abs. Cells were washed again and then incubated for 45 min each with the anti-CD40 5C3 Ab (200 ng/ml), polyclonal goat anti-ASM antiserum (1/100 dilution), anti-ceramide mAb 15B4 (1/30 dilution) or choleratoxin (10 µg/ml), respectively. The anti-CD40 5C3 Ab was visualized with a cyanine 3.18 (Cy3)-coupled anti-mouse Ig Ab, the anti-ASM Ab was displayed with a cyanine 5.18 (Cy5)-labeled anti-goat Ig Ab, the anti-ceramide Ab was visualized with a Cy5-labeled anti-mouse IgM, and choleratoxin was displayed with a Cy5-labeled anti-choleratoxin Ab. Control stainings were performed with irrelevant anti-mouse Ig mAbs or with pre-bled goat antiserum. Double stainings were performed with subsequent application of the indicated primary and secondary Abs. Fluorescence staining was viewed with a Nikon fluorescence microscope (Melville, NY) or a Leica TCS NT scanning confocal microscope (Munich, Germany). Fluorescence was analyzed by the simultaneous scanning of the two appropriate channels. Control samples were scanned separately and overlapping signals between different channels were compensated. Experiments using isotyped irrelevant primary Abs and the corresponding fluorescence-labeled secondary Abs were negative and confirmed the specificity of the results. Clustering was defined as one or several intense spots of fluorescence on the cell surface, whereas unstimulated cells displayed a homogenous distribution of the fluorescence throughout the membrane. In each experiment, the presence or absence of clustering in samples of 200 cells was scored by two independent observers. The results are given as the percentage of cells showing a cluster after the indicated time. All experiments were also performed without fixation in paraformaldehyde and revealed identical results.
Flow cytometry (FACS)
Cells in suspension were stained as described above using Cy3-labeled anti-ASM, Cy3-labeled anti-Myc-9E10 (1 µg/ml; Roche), Cy3-labeled anti-VSV (1 µg/ml; Roche) or Cy3-labeled anti-ceramide 15B4 (1/30 dilution), or isotype-matched irrelevant control mAbs and analyzed on a FACSCaliber (BD Biosciences, Franklin Lakes, NJ).
ASM activity
ASM activity was determined, as previously described (44, 45). In brief, cells were lysed in 50 mM of Tris (pH 7.4), 10 mM of bacitracin, 1 mM of benzamidine, 1 mM of Na3VO4, 10 µg/ml aprotinin and leupeptin (A/L), 0.1 mg/ml soybean trypsin inhibitor, and 0.2% Triton X-100. They were then sonicated three times for 10 s each and the lysates were centrifuged for 2 min at 600 x g. ASM lysis buffer that comprised equal amounts of 50 mM of Tris (pH 7.4), 3% Nonidet P-40, 1% Triton X-100, 1 mM of Na3VO4, and 100 µg/ml A/L was added and ASM was immunoprecipitated by using goat anti-ASM Abs for 4 h at 4°C. Immunocomplexes were immobilized using protein A/G-coupled agarose (Santa Cruz Biotechnology, Heidelberg, Germany), washed three times with ASM lysis buffer, and then washed three times with 50 mM of sodium acetate (pH 5.0), 0.2% Triton X-100, 1 mM of Na3VO4, and 10 µg/ml A/L. The complexes were then incubated for 30 min at 37°C with [14C]SM (0.5 µCi/sample, 54.5 mCi/mmol; NEN, Boston, MA) in 250 mM of sodium acetate (pH 5.0), 1.3 mM of EDTA, and 0.05% Nonidet P-40. Samples were extracted with a mixture of CHCl3, CH3OH (dilution, 2/1), and H2O. The upper phase was collected and radioactivity reflecting the degradation of [14C]SM was determined by liquid scintillation counting.
To analyze ASM activity on the cell surface, we stimulated 107 cells via CD40 ligation. We used a kit to biotinylate the cells according to the manufacturer’s instructions (Pierce, Rockford, IL). The cells were washed and lysed in 50 mM of Tris (pH 7.4), 1% Triton X-100, 1 mM of Na3VO4, and 10 µg/ml A/L. The lysates were subjected to precipitation with streptavidin-coupled agarose and the streptavidin precipitates were then analyzed for ASM activity, as described above.
Ceramide release
Cells were labeled for 48 h with 2 µCi/ml [3H]serine (27 mCi/mmol; NEN). Cells were then stimulated via CD40 ligation and ceramide was extracted in a solution of CHCl3, CH3OH, H2O, and pyridine (dilution, 60/160/6/1). The lower phase was collected and dried, and the phospholipids contained therein were degraded with methanolic NaOH at 37°C for 2 h. Samples were re-extracted in a solution of CHCl3, CH3OH, and 1 M of HCl (dilution, 100/100/1), dried, and resuspended in a solution of CHCl3 and CH3OH (dilution, 95/5). Lipids were separated on silica G60 thin-layer chromatography plates (Merck, Whitehouse Station, NJ) by using a solution of CHCl3, CH3OH, and CaCl2 (dilution, 65/35/8) and the substance that migrated to the same position as the radioactive [14C16]ceramide standard was identified as ceramide. Results obtained with [3H]serine labeling were confirmed with the 1,2-diacylglycerol-kinase method (not shown).
IL-12 assays
JY, ASM, or control-transfected NPDA cells were cultured in RPMI
1640 supplemented with 1% FCS and stimulated via CD40 using 100 ng/ml
plate-bound anti-CD40 for the indicated time. Nystatin, filipin,
-cyclodextrin, or -cyclodextrin were added to JY cells 30 min
before stimulation via CD40. Supernatants were collected and the
release of IL-12 was determined by ELISA exactly following the
instruction of the vendor (R&D Systems, Minneapolis, MN).
JNK activity
JY cells and ASM-deficient or reconstituted NPDA lymphocytes
were stimulated for 20 min, lysed in 25 mM of HEPES (pH 7.4), 0.1%
SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 125 mM of NaCl, 10 mM
each of NaF, Na3VO4, and
sodium pyrophosphate and 10 µg/ml A/L. JNK was immunoprecipitated
from the postcentrifugation lysates for 4 h at 4°C using
polyclonal rabbit anti-human JNK antiserum (Santa Cruz
Biotechnology). Immunocomplexes were immobilized on protein A/G (Santa
Cruz Biotechnology), incubated for an additional 60 min at 4°C,
washed twice in lysis buffer, twice in 132 mM of NaCl, 20 mM of HEPES,
5 mM of KCl, 1 mM of CaCl2, 0.7 mM of
MgCl2, 0.8 mM of MgSO4, 1%
Nonidet P-40, 2 mM of
Na3VO4, once in 100 mM of
Tris (pH 7.5), 0.5 M of LiCl, and, finally, twice in kinase buffer
consisting of 12.5 mM of MOPS (pH 7.5), 12.5 mM of
-glycerophosphate, 0.5 mM of EGTA, 7.5 mM of
MgCl2, 0.5 mM of NaF, 0.5 mM of
Na3VO4. Samples were
finally resuspended in kinase buffer supplemented with 10 µCi/sample
[
-32P]ATP (6000 Ci/mmol; NEN; DuPont
Pharmaceuticals, Wilmington, DE), 10 µM of ATP, and 1 µg/ml
GST-c-Jun (aa 1–79), incubated at 30°C for 15 min and the kinase
reaction was stopped by addition of 5 µl of boiling 5x reducing SDS
sample buffer. Samples were separated by 10% SDS-PAGE andanalyzed
by autoradiography. The substrate GST-c-Jun was expressed in
DH5-bacteria and purified via glutathione agarose.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether stimulation via CD40 ligation results
in clustering of the receptor, we stimulated JY B cells by using
cross-linked anti-CD40 Abs. Small clusters of CD40 formed on the
cell surface within a few seconds (Fig. 1
, A and B). Within
minutes of stimulation, these clusters enlarged and formed cap-like
structures. The specificity of this clustering process is evident,
because stimulation via CD40 ligation induced exclusive clustering of
the cognate receptor (Fig. 1, C and D), but not
of other receptors, including CD95, BCR, L-selectin, or
2 integrin. Stimulation with a very high
concentration of anti-CD40 (2 and 20 µg/ml secondary
F(ab')2) resulted in the formation of
cluster-like structures on almost all cells (92 ± 6%).
Clustering of CD40 occurred in choleratoxin-positive rafts (Fig. 1E). Choleratoxin binds to GM1 gangliosides highly
enriched in sphingolipid-rich rafts suggesting that CD40 clusters in
those rafts.
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2-fold upon CD40 ligation (Fig. 2A) correlating with a release
of ceramide (Fig. 2B).
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C). ASM deficiency did not
result in a general defect of receptor clustering, because clustering
of L-selectin, which does not activate ASM (46),
occurred in the NPDA/ASM-/- cells and the
NPDA/ASM+/+ cells (Fig. 2C).
To further investigate the role of ceramide in receptor clustering, we
tested CD40 clustering in AC-deficient B cells. AC metabolizes ceramide
and thus may function as a negative regulator of ceramide activity.
AC-deficient cells showed a marked increase in CD40 clustering (Fig. 2D); the level of clustering in AC-deficient cells
transfected with the AC expression vector was similar to that seen in
control B cells. These results indicate a role for ceramide in CD40
clustering, however, they do not exclude that some ceramide metabolites
are also involved in CD40 clustering.
CD40 stimulation induces translocation of ASM onto the extracellular leaflet of the cell membrane
To gain insight into the mechanisms of ASM-mediated CD40
clustering, we investigated the subcellular distribution of ASM and
whether ASM colocalizes with clustered CD40. FACS studies demonstrated
a translocation of ASM from an intracellular location in unstimulated
cells onto the cell surface after CD40-mediated stimulation (Fig. 3A). Permeabilization of the
cells with 0.1% Triton X-100 reveals that 10–15% of the cellular ASM
translocates to the cell surface upon stimulation. The specificity of
our detection of ASM was demonstrated by transfection of Myc-tagged ASM
and staining with Cy3-labeled anti-Myc 9E10 Ab and by transfection
with VSV-tagged ASM and staining with anti-VSV P5D4 Ab (Fig. 3B). To further confirm the translocation of functional ASM,
we biotinylated surface proteins and measured ASM activity in
streptavidin-selected immunoprecipitates. These studies revealed high
ASM activity on the surface of stimulated cells but not of unstimulated
cells (Fig. 3C).
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D). Together, these results indicate that activated CD40 clusters and colocalizes with ASM, which translocates onto the extracellular surface of the plasma membrane upon stimulation.
Surface ceramide triggers clustering of activated CD40
On the basis of our results, we hypothesized that ASM generates
extracellularly oriented ceramide upon stimulation of CD40. To test
this hypothesis, we stained JY B lymphocytes before or after
stimulation of CD40 with Cy3-labeled anti-ceramide 15B4 Ab. FACS
showed that a substantial amount of the anti-ceramide Ab bound to
the stimulated cells but not to unstimulated cells (Fig. 4A). CD40 and ceramide
colocalized in the cluster-like structure upon stimulation of the cells
via CD40 (Fig. 4B). Unstimulated cells almost completely
lacked surface ceramide. The importance of extracellularly oriented
ceramide for CD40 clustering is indicated by the abrogation of CD40
clustering upon neutralization of surface ceramide by the
anti-ceramide 15B4 mAb (Fig. 4C) or the destruction of
sphingolipid-rich rafts by -cyclodextrin, nystatin, or filipin (Fig. 4D). The inactive stereoisomer -cyclodextrin did not
affect CD40 clustering. Binding of the Ab to ceramide very likely
alters the biophysical properties, e.g. the mobility, of the lipid
and/or the ability of ceramide to interact with other lipids or
proteins and, thus, the Ab is able to prevent the formation of receptor
clusters.
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).
Combinations of noncross-linked 5C3 Ab and pure ASM or the Ab and
natural C16-ceramide were sufficient to initiate
CD40 clustering in NPDA/ASM+ cells and to restore
clustering in NPDA/ASM- cells (Fig. 5).
Cross-linking of 100 ng/ml anti-CD40 5C3 Ab by a secondary
F(ab')2 (1 µg/ml) also initiated CD40
clustering in ASM-expressing cells, but not in ASM-deficient B
lymphocytes (Fig. 5). The addition of the 5C3 Ab, pure ASM, or
C16-ceramide alone did not induce CD40
clustering. Likewise, CD40 clustering was not induced by treatment of
the cells with 5C3 anti-CD40 Abs and any of the following agents:
arachidonic acid (C20:4), SM,
dihydro-C2-ceramide, or
dihydro-C16-ceramide. A very high dose of
anti-CD40 Abs (2 µg/ml + 20 µg/ml secondary
F(ab')2) restored clustering of CD40 (Fig. 5) in
ASM-deficient cells, which is very likely due to the forced formation
of CD40 cluster by Ab aggregates.
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ASM-mediated CD40 clustering is required for CD40 signaling
We tested whether the activation of signaling events depends on
clustering of CD40. Genetic deficiency of ASM, treatment with
anti-ceramide Abs or destruction of rafts prevented CD40-mediated
release of IL-12 (Fig. 6, A
and B). These findings indicate that CD40 clustering in
ceramide-rich rafts is important for CD40-mediated cellular signaling.
The failure of ASM-deficient cells to release IL-12 was dose-dependent
and high doses of plate-bound anti-CD40 Abs restored, at least
partially, IL-12 release (Fig. 6C). Treatment of the cells
with -cyclodextrin, nystatin, or filipin did not affect surface
translocation of the ASM (Fig. 6D) or activation (not shown)
by CD40 indicating that signals occurring before CD40 clustering are
not altered by the drugs.
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-cyclodextrin or the inactive isomer
-cyclodextrin. ASM-deficient cells failed to activate JNK upon
stimulation via CD40, while retransfection of ASM restored JNK
activation (Fig. 6E). Likewise, treatment of the cells with
-cyclodextrin abrogated CD40-mediated JNK activation, whereas
-cyclodextrin was without effect. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data suggest the following model of ASM-mediated clustering of CD40: primary stimulation via CD40, which seems to be insufficient to trigger complete cellular activation, induces an activation and membrane translocation of the ASM from an intracellular compartment, most likely vesicles, to the cell membrane. Fusion of ASM-containing vesicles with the cell membrane results in exposure of the ASM to the extracellular surface. ASM causes a localized release of extracellularly oriented ceramide and this molecule may function in several ways in the clustering process. First, ceramide has been shown to self-aggregate (48, 49, 50). A self-aggregation of ceramide in pre-existing sphingolipid-rich rafts may generate very small primary signaling microdomains. Second, because ceramide-rich microdomains spontaneously fuse (51), the generation of ceramide in those sphingolipid-rich rafts may trigger the fusion of those very small entities into larger membrane domains. The fusion of very small microdomains triggers the clustering of CD40 molecules already present in those rafts. Previous studies on dendritic cells and B lymphocytes (5, 6), as well as preliminary data from our lab, indicate a substantial amount of CD40 in Triton X-100 insoluble membrane fractions already present before receptor stimulation suggesting that ceramide-mediated fusion of small rafts to larger platforms constitutes a major mechanism to cluster CD40. In addition, ceramide may also gather CD40 molecules into membrane rafts, prevent diffusion of CD40 molecules from rafts, or function in both ways. The findings from our studies on AC-deficient Farber lymphocytes as well as those from studies that included anti-ceramide 15B4 Ab binding strongly support a crucial role of ceramide in CD40 clustering. However, we cannot exclude the possibility that some metabolite(s) of ceramide, e.g., glycosylated ceramides, are involved in clustering, because some ceramide metabolites may also accumulate in Farber lymphocytes upon stimulation and may be detected by the anti-ceramide Abs. The FACS analysis and fluorescence microscopy examinations presented in this study revealed that the anti-ceramide Ab 15B4 did not bind to unstimulated cells, whereas binding occurred rapidly after stimulation of CD40. In addition, ASM-deficient cells failed to bind the Ab even after stimulation with anti-CD40 Ab. This result excludes the possibility of a substantive reaction with cholesterol, SM, or phospholipids. The observation that loading intact cells with C16-ceramide resulted in substantial Ab binding to the cell surface provides additional evidence of a specific detection of ceramide. Also, the anti-ceramide Ab detected a single band on high performance thin layer chromatography plates containing lipid extracts from CD40-stimulated cells and this band migrated to the same position as C16-ceramide. The anti-ceramide Ab did not detect dihydro-C16-ceramide or SM (E. Gulbins, unpublished data).
In the present study, we used several approaches to determine ASM translocation onto the cell surface; the specificity of these findings was confirmed by Ab staining of cells transfected with Myc- or VSV-tagged ASM constructs. In addition, the anti-ASM Ab failed to bind to B lymphocytes derived from ASM knockout mice, whereas B lymphocytes from normal control mice showed a rapid surface translocation of ASM upon CD40 ligation (E. Gulbins, unpublished observations).
Clustering of CD40 in rafts may serve many functions. First, multimerization of CD40 may permit the transactivation of molecules associated with CD40, e.g., JAK3 (34), even if only a few dispersed molecules are primarily active. Second, translocation of CD40 may bring the receptor into close contact with signaling molecules present in rafts and may permit activation of these signaling molecules by CD40. Third, inhibitory signaling molecules, such as the tyrosine phosphatase CD45 (4, 22), might be excluded from rafts, a situation that may further enhance signaling via CD40. Fourth, immobilization of CD40 in a defined membrane domain may stabilize the interaction of CD40 with its ligand. Lateral diffusion of raft-trapped CD40 might be greatly reduced; thus, the presence of CD40 in rafts may prolong binding to its ligand. Fifth, the translocation of CD40 into ceramide-enriched sphingolipid-rich rafts may promote interaction with and synergistic cell activation by other receptors, in particular, the BCR. Studies have shown that clustering of the BCR in membrane rafts is essential for its stimulatory functions (4, 19); however, whether CD40 and the BCR cluster in the same rafts remains unknown. Finally, the generation of ceramide in rafts may directly alter CD40 signaling, as recently shown for ceramide-dependent recruitment of caveolin 1 to receptor complexes in rafts, an activity that inhibits phosphatidylinositol 3-kinase (52).
In conclusion, CD40 ligation induces activation and surface translocation of ASM, which colocalizes with sphingolipid-rich membrane rafts. The activity of ASM causes the localized release of extracellularly oriented ceramide in sphingolipid-rich rafts, which in turn mediates clustering of CD40. Finally, CD40 clustering in ceramide-enriched rafts is required for the initiation of signaling within cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Erich Gulbins, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794. E-mail address: erich.gulbins{at}stjude.org
3 Abbreviations used in this paper: BCR, B cell receptor; DIG, detergent insoluble glycosphingolipid-enriched membrane domain; ASM, acid sphingomyelinase; TRAF, TNFR-associated factor; JAK3, Janus family kinase 3; NPDA, Niemann-Pick disease type A; VSV, vesicular stomatitis virus; AC, acid ceramidase; SM, sphingomyelin; JNK, c-Jun N-terminal kinase; Cy3, fluorescent cyanine 3.18; Cy5, fluorescent cyanine 5.18; A/L, aprotinin and leupeptin.
Received for publication May 9, 2001. Accepted for publication October 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated recruitment of p53/p56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc. Natl. Acad. Sci. USA 92:9201.B by TNF receptor 2 and CD40. Science 269:1424.2. J. Exp. Med. 179:673.This article has been cited by other articles:
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  J. A. Rotolo, B. Stancevic, S. X. Lu, J. Zhang, D. Suh, C. G. King, L. W. Kappel, G. F. Murphy, C. Liu, Z. Fuks, et al. Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease Blood, October 22, 2009; 114(17): 3693 - 3706. [Abstract] [Full Text] [PDF]
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 Z. Li, Y. Li, M. Chakraborty, Y. Fan, H. H. Bui, D. A. Peake, M.-S. Kuo, X. Xiao, G. Cao, and X.-C. Jiang Liver-specific Deficiency of Serine Palmitoyltransferase Subunit 2 Decreases Plasma Sphingomyelin and Increases Apolipoprotein E Levels J. Biol. Chem., September 25, 2009; 284(39): 27010 - 27019. [Abstract] [Full Text] [PDF]
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 S. Uhlig and E. Gulbins Sphingolipids in the Lungs Am. J. Respir. Crit. Care Med., December 1, 2008; 178(11): 1100 - 1114. [Abstract] [Full Text] [PDF]
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C. Reyes-Moreno, E. Sharif-Askari, J. Girouard, C. Leveille, M. Jundi, A. Akoum, R. Lapointe, A. Darveau, and W. Mourad Requirement of Oxidation-dependent CD40 Homodimers for CD154/CD40 Bidirectional Signaling J. Biol. Chem., July 6, 2007; 282(27): 19473 - 19480. [Abstract] [Full Text] [PDF]
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 D. Pozo, M. Vales-Gomez, N. Mavaddat, S. C. Williamson, S. E. Chisholm, and H. Reyburn CD161 (Human NKR-P1A) Signaling in NK Cells Involves the Activation of Acid Sphingomyelinase J. Immunol., February 15, 2006; 176(4): 2397 - 2406. [Abstract] [Full Text] [PDF]
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 E. Gulbins and P. L. Li Physiological and pathophysiological aspects of ceramide Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R11 - R26. [Abstract] [Full Text] [PDF]
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 A. Y. Zhang, F. Yi, G. Zhang, E. Gulbins, and P.-L. Li Lipid Raft Clustering and Redox Signaling Platform Formation in Coronary Arterial Endothelial Cells Hypertension, January 1, 2006; 47(1): 74 - 80. [Abstract] [Full Text] [PDF]
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 J. Wu, Y. Cheng, B. A. G. Jonsson, A. Nilsson, and R.-D. Duan Acid sphingomyelinase is induced by butyrate but does not initiate the anticancer effect of butyrate in HT29 and HepG2 cells J. Lipid Res., September 1, 2005; 46(9): 1944 - 1952. [Abstract] [Full Text] [PDF]
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H. Grassme, A. Riehle, B. Wilker, and E. Gulbins Rhinoviruses Infect Human Epithelial Cells via Ceramide-enriched Membrane Platforms J. Biol. Chem., July 15, 2005; 280(28): 26256 - 26262. [Abstract] [Full Text] [PDF]
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J. A. Rotolo, J. Zhang, M. Donepudi, H. Lee, Z. Fuks, and R. Kolesnick Caspase-dependent and -independent Activation of Acid Sphingomyelinase Signaling J. Biol. Chem., July 15, 2005; 280(28): 26425 - 26434. [Abstract] [Full Text] [PDF]
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 P. A. Lang, D. S. Kempe, V. Tanneur, K. Eisele, B. A. Klarl, S. Myssina, V. Jendrossek, S. Ishii, T. Shimizu, M. Waidmann, et al. Stimulation of erythrocyte ceramide formation by platelet-activating factor J. Cell Sci., March 15, 2005; 118(6): 1233 - 1243. [Abstract] [Full Text] [PDF]
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 M. Czarny and J. E. Schnitzer Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1344 - H1352. [Abstract] [Full Text] [PDF]
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A. B. Abdel Shakor, K. Kwiatkowska, and A. Sobota Cell Surface Ceramide Generation Precedes and Controls Fc{gamma}RII Clustering and Phosphorylation in Rafts J. Biol. Chem., August 27, 2004; 279(35): 36778 - 36787. [Abstract] [Full Text] [PDF]
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C. Bezombes, S. Grazide, C. Garret, C. Fabre, A. Quillet-Mary, S. Muller, J.-P. Jaffrezou, and G. Laurent Rituximab antiproliferative effect in B-lymphoma cells is associated with acid-sphingomyelinase activation in raft microdomains Blood, August 15, 2004; 104(4): 1166 - 1173. [Abstract] [Full Text] [PDF]
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 S. Lacour, A. Hammann, S. Grazide, D. Lagadic-Gossmann, A. Athias, O. Sergent, G. Laurent, P. Gambert, E. Solary, and M.-T. Dimanche-Boitrel Cisplatin-Induced CD95 Redistribution into Membrane Lipid Rafts of HT29 Human Colon Cancer Cells Cancer Res., May 15, 2004; 64(10): 3593 - 3598. [Abstract] [Full Text] [PDF]
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S. Gupta, R. Natarajan, S. G. Payne, E. J. Studer, S. Spiegel, P. Dent, and P. B. Hylemon Deoxycholic Acid Activates the c-Jun N-terminal Kinase Pathway via FAS Receptor Activation in Primary Hepatocytes: ROLE OF ACIDIC SPHINGOMYELINASE-MEDIATED CERAMIDE GENERATION IN FAS RECEPTOR ACTIVATION J. Biol. Chem., February 13, 2004; 279(7): 5821 - 5828. [Abstract] [Full Text] [PDF]
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F.-X. Contreras, A.-V. Villar, A. Alonso, R. N. Kolesnick, and F. M. Goni Sphingomyelinase Activity Causes Transbilayer Lipid Translocation in Model and Cell Membranes J. Biol. Chem., September 26, 2003; 278(39): 37169 - 37174. [Abstract] [Full Text] [PDF]
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C. Contin, V. Pitard, T. Itai, S. Nagata, J.-F. Moreau, and J. Dechanet-Merville Membrane-anchored CD40 Is Processed by the Tumor Necrosis Factor-{alpha}-converting Enzyme: IMPLICATIONS FOR CD40 SIGNALING J. Biol. Chem., August 29, 2003; 278(35): 32801 - 32809. [Abstract] [Full Text] [PDF]
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W. Drobnik, G. Liebisch, F.-X. Audebert, D. Frohlich, T. Gluck, P. Vogel, G. Rothe, and G. Schmitz Plasma ceramide and lysophosphatidylcholine inversely correlate with mortality in sepsis patients J. Lipid Res., April 1, 2003; 44(4): 754 - 761. [Abstract] [Full Text] [PDF]
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A. Krippner-Heidenreich, F. Tubing, S. Bryde, S. Willi, G. Zimmermann, and P. Scheurich Control of Receptor-induced Signaling Complex Formation by the Kinetics of Ligand/Receptor Interaction J. Biol. Chem., November 8, 2002; 277(46): 44155 - 44163. [Abstract] [Full Text] [PDF]
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H. Grassme, J. Bock, J. Kun, and E. Gulbins Clustering of CD40 Ligand Is Required to Form a Functional Contact with CD40 J. Biol. Chem., August 9, 2002; 277(33): 30289 - 30299. [Abstract] [Full Text] [PDF]
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V. Ayllon, A. Fleischer, X. Cayla, A. Garcia, and A. Rebollo Segregation of Bad from Lipid Rafts Is Implicated in the Induction of Apoptosis J. Immunol., April 1, 2002; 168(7): 3387 - 3393. [Abstract] [Full Text] [PDF]
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