By
From the * Department of Immunology, Istituto Superiore di Sanità, 00161 Rome; and Department
of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, 00133 Rome, Italy
Ceramides are intramembrane diffusible mediators involved in transducing signals originated
from a variety of cell surface receptors. Different adaptive and differentiative cellular responses, including apoptotic cell death, use ceramide-mediated pathways as an essential part of the program. Here, we show that human dendritic cells respond to CD40 ligand, as well as to tumor
necrosis factor- and IL-1
, with intracellular ceramide accumulation, as they are induced to
differentiate. Dendritic cells down-modulate their capacity to take up soluble antigens in response to exogenously added or endogenously produced ceramides. This is followed by an impairment in presenting soluble antigens to specific T cell clones, while cell viability and the capacity to stimulate allogeneic responses or to present immunogenic peptides is fully preserved. Thus, ceramide-mediated pathways initiated by different cytokines can actively modulate professional antigen-presenting cell function and antigen-specific immune responses.
Dendritic cells (DCs) represent key players in the immune response (1). They capture and process antigens
in nonlymphoid tissues, then move into T cell-dependent
areas of secondary lymphoid organs to prime naive T cells
and initiate the immune response (2). Along this process,
DCs lose antigen-capturing ability as they differentiate into
mature, fully stimulatory antigen-presenting cells (APCs)
(3). The recent establishment of an in vitro system that allows human DCs to be mantained in culture preserving
their immature phenotype, i.e., efficient antigen uptake
and processing, has revealed a unique tool to gain insights
into the basic mechanisms governing the differentiation of
DCs (4). Evidence has been provided, in fact, that tumor
necrosis-factor- Sphingomyelin breakdown by sphingomyelinases, with
resulting ceramide release, is a major signaling event that
follows both TNF-R1 and IL-1 In Vitro Culture of Human DCs.
PBMCs obtained by standard
Ficoll-Paque method (Organon Teknika, Durham, NC) were
separated on multistep Percoll gradients (Pharmacia Fine Chemicals, Uppsala, Sweden) and the light density fraction from the
42.5-50% interface was recovered and depleted of CD19+ and
CD2+ cells using magnetic beads coated with specific antibodies
(Dynal, Oslo, Norway). The remaining cells were cultured in
RPMI-1640 supplemented with 2 mM L-glutamine, 1% nonessential aminoacids, 1% pyruvate, 50 µg/ml kanamicin, 5 × 10 Ceramide Mass Measurement Assay.
After stimulation, lipids were
extracted and then incubated with Escherichia coli diacylglycerol
kinase. Ceramide phosphate was then isolated by TLC using
CHCl3/CH3OH/CH3COOH (65:15:5) as solvent (13, 14). Authentic ceramide-1-phosphate was identified by autoradiography
at Rf 0.25. Quantitative results for ceramide production are expressed as pmol ceramide-1-phosphate/106 cells.
Endocytosis and Antigen Presentation Assays.
2 × 105 DCs were
resuspended in 200 µl RPMI buffered with 25 mM Hepes + 10%
FCS. C2-ceramide and C2-dihydroceramide (Sigma Immunochemicals, St. Louis, MO) were reconstituted in EtOH at 15 mM
and stored at (TNF-
) (via the p55 TNF-R1), IL-1
,
CD40 ligand (CD40L), and bacterial lipopolysaccaride (LPS)
can promote DC differentiation in vitro, resulting in irreversible structural and functional changes associated with a
mature DC phenotype, including downregulation of antigen uptake and processing capacity (5). However, little is
known about the intracellular signals that are responsible for
mediating these changes in DCs after cytokines or bacterial
products exposure.
receptor engagement by
their respective ligands (6, 7). Ceramide diffuses within membranes activating downstream effectors, including different
protein kinases and phosphatases, eventually leading to a
variety of adaptive and differentiative cellular responses (8).
Intriguingly, LPS, also a potent inducer of DC differentiation (5), mimics many of the cellular responses initiated by
TNF-
and IL-1
, without inducing sphingomyelin hydrolysis but likely becouse of its structural analogy with ceramide itself (11, 12). Therefore, we investigated whether some
of the differentiative changes induced in DCs by inflamatory cytokines and LPS could be mediated by ceramides.
5
M 2-ME (GIBCO BRL, Gaithersburg, MD) + 10% FCS (Hyclone Laboratories, Inc., Logan, UT) in the presence of 50 ng/ml
GM-CSF and 1000 U/ml IL-4 (provided by Dr. A. Lanzavecchia, Basel Institute for Immunology, Switzerland). Cultured
DCs were routinely >90% CD1+, HLA-DR+, CD14
, and were
used after 5-7 d of culture.
20°C until use. Diacylglycerol was purchased from Amersham (Buckingham, England). Lucifer yellow (LY),
FITC-dextran (DX) (Molecular Probes, Inc., Eugene, OR), or
HRP (Sigma Immunochemicals) were reconstituted in PBS,
stored at 4°C, and spun in a microfuge before use to eliminate aggregates. To quantify LY and FITC-DX, cells were washed four
times with cold PBS containing 1% FCS and 0.01% NaN3 and
analyzed on a FACScan® (Becton Dickinson, Mountain View, CA),
using propidium iodide to exclude dead cells. For horseradish
peroxidase (HRP) quantification, cells were washed four times as
above then four times with PBS alone with one tube change, lysed
with 0.05% Triton X-100 in 10 mM Tris buffer pH 7.4 for 30 min, and the enzyme activity of the lysate was measured using
O-phenilendiamine and H2O2 as substrates with reference to a
standard curve. Tetanus toxoid (TT)-specific T cell clones KS140
and KB24 and TT peptide P2 (residues 830-843) were provided
by Dr. A. Lanzavecchia. TT antigen was purchased from Connaught (Ontario, Canada).
DNA Labeling and Flow Cytometry Analysis. 2 × 105 DCs were treated in 200 µl RPMI 10% FCS with 80 µM C2-ceramide for 10 min on ice, then for 1 h at 37°C. After incubation, cells were washed and left in culture for 48 h. Cells were then recovered and processed for propidium iodide staining and FACS® analysis as previously described (13).
Other Reagents.
Supernatant from J558L cells stably expressing a chimeric mCD40L-mCD8 construct, provided by Dr. P. Lane, (Basel Institute for Immunology) was used as a source of
CD40L. Recombinant human TNF-
with R32W and S86T
substitutions (TNF-R1 specific) and TNF-
with D143N and
A145R substitutions (TNF-R2 specific) were provided by Drs.
W. Lesslauer and H. Loetscher (Hoffman La Roche, Ltd., Basel,
Switzerland). IL-1
was provided by Dr. L. Melli (IRIS, Siena,
Italy).
IL-1 and TNF-
have been shown to induce transient ceramide accumulation in tumor cell lines
(16, 17). It was not known whether CD40, like other TNF
receptor family members such as TNF-R1 p55, Fas/APO-1,
or NGF-R p75 (13, 16, 18), also signaled through ceramide
generation. Therefore, we investigated whether cross-linking of CD40 was able to induce ceramide accumulation in
cultured immature DCs. Fig. 1 shows that CD40L, as well
as IL-1
and TNF-
, engaging TNF-R1 p55 but not
TNF-R2 p75, were all potent inducers of ceramide generation in cultured DCs. Because CD40L, IL-1
, and TNF-
trigger in vitro maturation of DCs (5), as does LPS, which
is structurally analogous to ceramide itself (11), these results
raised the possibility that a common ceramide-mediated pathway, mimicked by LPS, could be responsible for some
of the functional changes observed during in vitro maturation of DCs.
Ceramides Down-modulate Macromolecule Uptake by DCs.
To test this hypothesis directly, we investigated whether
exposure to exogenous cell-permeant C2-ceramide could
down-modulate DC antigen uptake ability. DCs capture
antigen either via macropinocytosis, a cytoskeleton-dependent type of fluid phase endocytosis initiated by membrane
ruffling and formation of large vesicles, or receptor-mediated endocytosis through Fc and mannose receptors (5).
As shown in Fig. 2, C2-ceramide could inhibit the uptake
of three different classical endocytosis markers and their
time-dependent accumulation into DCs. Both macropinocytosis, as assessed by LY and FITC-DX, and receptormediated endocytosis, as assessed by limiting amounts of
HRP, were significantly affected. Comparable results were
also obtained using C6-ceramide, a longer acyl chain ceramide analogue (data not shown). By contrast, C2-dihydroceramide, a structural analogue of C2-ceramide that lacks a
double bond at the 4-5 position in the sphingoid base, was
ineffective. Similarly, exposure to other diffusible signaltransducing lipid mediators such as diacylglycerol, did not
affect macromolecule uptake ability of DCs (Fig. 2, A, C, E).
We then tested whether the endogenous production of
ceramide would result in a similar inhibition of the endocytic
ability of DCs. Exposure of cultured DCs to exogenous
sphingomyelinase, which results in intracellular ceramide
accumulation (data not shown), also induced a dose-dependent inhibition of HRP uptake (Fig. 3 A) and substantially
retarded its time-dependent accumulation (Fig. 3 B). Taken
together, these results indicated that ceramide could specifically mediate inhibition of macromolecules uptake by DCs.
Ceramides Down-modulate Soluble Antigen Presentation by DCs.
Cultured DCs are extremely efficient at presenting
soluble antigen to specific T cells (2). In vitro maturation of
DCs promoted by short term exposure to TNF- results in
a severalfold decrease of the antigen presentation capacity,
associated with an increase in T cell stimulatory ability (4).
Therefore, we tested whether ceramide could be sufficient
for effectively modulating antigen presentation to T cells
by using two different soluble antigens, TT and a soluble
extract of P. judaica pollen (PjE). Cultured DCs were exposed to C2-ceramide and then pulsed with TT or PjE,
before being used to challenge antigen-specific T cell clones
(15, 19). Fig. 4 shows that C2-ceramide induced a ~50fold reduction in the ability of DCs to present PjE, and
~100-fold reduction in the ability to present TT to their
respective T cell clones (Fig. 4, A and B). By contrast, DCs
treated with C2-ceramide were at least as efficient as untreated DCs in presenting nonprocessed antigen, i.e., in
presenting an immunogenic TT peptide to the same TTspecific T cell clone (Fig 4 C). Ceramide analogue C2-dihydroceramide was ineffective in blocking the response to
soluble antigens (Figs. 4, A, B, and C).
C2-ceramide is known to induce apoptotic cell death when administered to hemopoietic tumor cell lines or to in vivoactivated primary lymphoid cells within 6-12 h (20). Therefore, we checked whether the observed changes in antigen-processing capacity were due to loss of cell viability. C2-ceramide-treated DCs cultured for as long as 48 h excluded Trypan blue, displayed normal morphology, and did not show any DNA fragmentation by propidium iodide staining and FACS® analysis (Fig. 4, D and E). Moreover, C2-ceramide treatment did not affect the ability of DCs to stimulate allogeneic T cells (Fig. 4 F).
Finally, we investigated whether the endogenous production of ceramide would affect the ability of DCs to present
soluble antigen to T cells. Cultured DCs were treated with
exogenous sphingomyelinase before being pulsed with TT,
or with a TT peptide, and used to challenge a TT-specific
T cell clone. Fig. 5 shows that endogenous ceramide production almost completely prevented presentation of soluble TT antigen, but had no inhibitory effect on TT peptide presentation by DCs, to the same TT-specific T cell clone.
In this paper, we provide evidence that ceramides inhibit
the antigen-capturing ability of cultured DCs, thereby suggesting a common molecular basis for CD40L, TNF-,
and IL-1
, or bacterial products such as LPS, to downmodulate antigen presentation by professional APC (5). In
fact, we show that CD40L, as well as TNF-
or IL-1
,
were all strong inducers of ceramide accumulation in DCs.
Ceramides may specifically control antigen capturing and processing by DCs, as other cytokine-mediated differentiation events, i.e., upregulation of LFA1, B7-1, ICAM-1,
and MHC molecules, were not consistently affected by ceramide exposure (data not shown). Accordingly, the enhanced immunostimulatory ability of mature DCs could
not be promoted by exogenous ceramides, suggesting that
additional intracellular mediators participate in the maturative process. Importantly, specific immunoefficiency of DCs
can be inhibited without affecting cell viability or the ability to present nonprocessed antigen.
A possible explanation for these findings may reside in the capacity of endogenously released ceramides to interfere with vesicular trafficking. In fact, ceramides have been shown to directly inhibit endocytosis (23) and glycoprotein transport through the Golgi complex in CHO cells (24). Perturbing anterograde transport through the Golgi may prevent newly synthesized MHC class II molecules to reach endosomal compartments to be loaded with peptides derived from hydrolyzed antigen. Interestingly, the fungal antibiotic brefeldin A (BFA), a classic inhibitor of both endogenous and exogenous antigen processing and presentation (25, 26), which causes disassembly of the Golgi apparatus (27) and its fusion with the ER and with early endosomes (28, 29), also triggers sphingomyelin hydrolysis resulting in ceramide production (30). Therefore, it is likely that the capacity of BFA to modulate antigen presentation is mediated by endogenously released ceramide.
Ceramides are emerging as intramembrane messengers involved in a variety of cellular adaptive and differentiative responses. Here, we provide evidence for a novel important function of ceramides in highly specialized cells such as DCs, which is modulation of soluble antigen presentation. Moreover, our data suggest that the capacity of ceramides to perturb intracellular membrane trafficking may be exploited by extracellular ligands able to trigger sphingomyelin hydrolysis, or by bacterial products that mimic ceramide, such as LPS, in order to regulate professional APC function and antigen-specific immune responses.
Address correspondence to Roberto Testi, Department of Experimental Medicine and Biochemical Sciences, University of Tor Vergata, via Tor Vergata 135, 00133 Rome, Italy.
Received for publication 13 September 1996
This work has been supported by Istituto Superiore di Sanitá (Progetto Tubercolosi), Associazione Nazionale Ricerca sul Cancro, CNR (Progetto Citochine), MURST, and European Community (Projects Human Capital and Mobility and Biomed 2). R. De Maria is an AIRC fellowship holder.We thank Drs. A. Lanzavecchia, P. Lane, W. Lesslauer, and H. Loetscher for reagents.
1. | Steinman, R.M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9: 271-296 [Medline] . |
2. | Austyn, J.M.. 1992. Antigen uptake and presentation by dendritic leukocytes. Semin. Immunol. 4: 227-236 [Medline] . |
3. | Austyn, J.M.. 1996. New insights into the mobilization and phagocytic activity of dendritic cells. J. Exp. Med. 183: 1287-1292 [Medline] . |
4. |
Sallusto, F., and
A. Lanzavecchia.
1994.
Efficient presentation
of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor
plus interleukin 4 and downregulated by tumor necrosis factor ![]() |
5. | Sallusto, F., M. Cella, C. Danieli, and A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182: 389-400 [Abstract] . |
6. | Kolesnick, R., and D.W. Golde. 1994. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling. Cell. 77: 325-328 [Medline] . |
7. | Heller, R.A., and M. Krönke. 1994. Tumor necrosis factor- mediated signaling pathways. J. Cell Biol. 126: 5-9 [Medline] . |
8. |
Hannun, Y.A..
1994.
The sphingomyelin cycle and second
messenger function of ceramide.
J. Biol. Chem.
269:
3125-3128
|
9. | Kolesnick, R., and Z. Fuks. 1995. Ceramide: a signal for apoptosis or mitogenesis? J. Exp. Med. 181: 1949-1952 [Medline] . |
10. | Testi, R. 1996. Sphingomyelin breakdown and cell fate. Trends Biochem. Sci. In press. |
11. |
Joseph, C.K.,
S.D. Wright,
W.G. Bornmann,
J.T. Randolph,
E.R. Kumar,
R. Bittman,
J. Liu, and
R.N. Kolesnick.
1994.
Bacterial lipopolysaccaride has structural similarity to ceramide
and stimulates ceramide-activated protein kinase in myeloid
cells.
J. Biol. Chem.
269:
17606-17610
|
12. | Wright, S., and R.N. Kolesnick. 1995. Does endotoxin stimulate cells by mimicking ceramide? Immunol. Today. 16: 297-302 [Medline] . |
13. | Cifone, M.G., R. De Maria, P. Roncaioli, M.R. Rippo, M. Azuma, L.L. Lanier, A. Santoni, and R. Testi. 1994. Apoptotic signaling through CD95 (Fas/APO-1) activates an acidic sphingomyelinase. J. Exp. Med. 180: 1547-1552 [Abstract] . |
14. | Cifone, M.G., P. Roncaioli, R. De Maria, G. Camarda, A. Santoni, G. Ruberti, and R. Testi. 1995. Multiple signaling originate at the Fas/Apo-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5859-5868 [Abstract] . |
15. |
Sallusto, F.,
S. Corinti,
C. Pini,
M.M. Biocca,
G. Bruno, and
G. Di Felice.
1996.
Parietaria judaica-specific T cell clones
from atopic patients: heterogeneity in restriction, V![]() |
16. |
Kim, M.-Y.,
C. Linardic,
L. Obeid, and
Y. Hannun.
1991.
Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor ![]() ![]() |
17. |
Mathias, S.,
A. Younes,
C.-C. Kan,
I. Orlow,
C. Joseph, and
R.N. Kolesnick.
1993.
Activation of the sphingomyelin signaling pathway in intact EL4 cells and in a cell-free system by
IL-1![]() |
18. | Dobrowsky, R.T., M.H. Werner, A.M. Castellino, M.V. Chao, and Y.A. Hannun. 1994. Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor. Science (Wash. DC). 265: 1596-1599 [Medline] . |
19. | Valitutti, S., S. Muller, M. Cella, E. Padovan, and A. Lanzavecchia. 1995. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature (Lond.). 375: 148-151 [Medline] . |
20. | Obeid, L.M., C.M. Linardic, L.A. Karolak, and Y.A. Hannun. 1993. Programmed cell death induced by ceramide. Science (Wash. DC). 259: 1769-1771 [Medline] . |
21. | Jarvis, W.D., R.N. Kolesnick, F.A. Fornari, R.S. Traylor, D.A. Gerwitz, and S. Grant. 1994. Induction of apoptotic damage and cell death by activation of the sphingomyelin pathway. Proc. Natl. Acad. Sci. USA. 91: 73-77 [Abstract] . |
22. |
De Maria, R.,
M. Boirivant,
M.G. Cifone,
P. Roncaioli,
M. Hahne,
J. Tschopp,
F. Pallone,
A. Santoni, and
R. Testi.
1996.
Functional expression of Fas and Fas ligand on human
gut lamina propria lymphocytes. A potential role for the
acidic sphingomyelinase pathway in normal immunoregulation.
J. Clin. Invest.
97:
316-322
|
23. |
Chen, C.-S.,
A.G. Rosenwald, and
R.E. Pagano.
1995.
Ceramide as a modulator of endocytosis.
J. Biol. Chem.
270:
13291-13297
|
24. | Rosenwald, A.G., and R.E. Pagano. 1993. Intracellular transport of ceramide and its metabolites at the Golgi complex: insights from short-chain analogs. Adv. Lipid Res. 26: 101-118 [Medline] . |
25. | Yewdell, J.W., and J.R. Bennink. 1989. Brefeldin A specifically inhibits presentation of protein antigens to cytotoxic T lymphocytes. Science (Wash. DC). 244: 1072-1075 [Medline] . |
26. | Adorini, L., S.J. Ullrich, E. Appella, and S. Fuchs. 1990. Inhibition by brefeldin A of presentation of exogenous protein antigens to MHC class II-restricted T cells. Nature (Lond.). 346: 63-66 [Medline] . |
27. |
Fujiwara, T.,
K. Oda,
S. Yokota,
A. Takatsuki, and
Y. Ikehara.
1988.
Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the endoplasmic reticulum.
J. Biol. Chem.
263:
18545-18552
|
28. | Lippincott-Schwartz, J., L.C. Yuan, J.S. Bonifacino, and R.D. Klausner. 1989. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 56: 801-813 [Medline] . |
29. | Wood, S.A., J.E. Park, and W.J. Brown. 1991. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 67: 591-600 [Medline] . |
30. |
Linardic, C.M.,
S. Jayadev, and
Y.A. Hannun.
1992.
Brefeldin A promotes hydrolysis of sphingomyelin.
J. Biol. Chem.
267:
14909-14911
|
31. | Lane, P., T. Brocker, S. Hubele, E. Padovan, A. Lanzavecchia, and F. McConnell. 1993. Soluble CD40 ligand can replace the normal T cell-derived CD40 ligand signal to B cells in T cell-dependent activation. J. Exp. Med. 177: 1209-1213 [Abstract] . |
32. |
Mackay, F.,
H. Loetscher,
D. Stueber,
G. Gehr, and
W. Lesslauer.
1993.
Tumor necrosis factor ![]() ![]() |