Inhibitory activity of a ceramide library on interleukin-4 production from activated T cells

Article abstract of DOI:10.1016/j.bmc.2005.01.027

Allergic diseases are hypersensitivity disorders associated with the production of specific immunoglobulin E (IgE) to environmental allergens. Interleukin (IL)-4, produced primarily by CD4⁺ T cells, is an important stimulus for the switch of th

Full text of DOI:10.1016/j.bmc.2005.01.027

Bioorganic & Medicinal Chemistry 13 (2005) 2589–2595
Inhibitory activity of a ceramide library on interleukin-4
production from activated T cells
Jin Park,a,b Qian Li,c Young-Tae Changc and Tae Sung Kima,b,
*
aDepartment of Pharmacy, College of Pharmacy, Chonnam National University, Gwangju 500-757, Republic of Korea
bResearch Institute of Drug Development, Chonnam National University, Gwangju 500-757, Republic of Korea
cDepartment of Chemistry, New York University, New York, NY 10003, USA
Received 13 November 2004; revised 17 January 2005; accepted 18 January 2005
Abstract—Allergic diseases are hypersensitivity disorders associated with the production of specific immunoglobulin E (IgE) to envi-
ronmental allergens. Interleukin (IL)-4, produced primarily by CD4+ T cells, is an important stimulus for the switch of the antibody
isotype to IgE in both mice and humans. In this study we investigated the inhibitory activity of IL-4 production in activated T cells
by screening ceramide derivatives prepared by solid phase combinatorial chemistry. Many ceramide derivatives significantly inhib-
ited IL-4 production in T cells. In particular, ceramide derivatives with a lauroyl group showed strong inhibitory activities on IL-4
production in both phorbol 12-myristate 13-acetate (PMA)-activated EL4 T cells and antigen-primed cells, suggesting that they can
be used as compounds for the development of anti-allergic agents.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Previous studies have showed that the glycosphingolipids
derived from tumours or parasites exhibited immunosup-
pressive activity.8 A role for ceramides in regulating the
immune response was suggested based on several obser-
vations, including its role in apoptosis, TCR expression,
T cell proliferation, T cell activation and cytokine expres-
sion.9 Because ceramides play a diversity role in regula-
tion of immune responses and immunosuppressive
activity, especially T cell responses,10 we synthesize a cer-
amide library and investigate their inhibitory activities on
IL-4 production concerned with allergic inflammation.
Ceramide refers to a family of highly hydrophobic mole-
cules that contain a variable length fatty acid linked to
sphingosine or a related long chain base.1 Ceramide
has a number of important physiologic functions that
regulate cellular homeostasis including regulation of
the stress response, induction of cell differentiation, reg-
ulation of cell cycle arrest and apoptosis.2
IL-4, a pleiotropic cytokine produced by activated T
cells, basophils and mast cells, regulates many cellular
and humoural immune responses. Dysregulation of IL-
4 expression results in uncontrolled allergic inflamma-
tion including asthma and aberrant immune responses
to pathogens.3 IL-4 has a variety of other effects in
hepatopoietic tissues. It increases the expression of class
II MHC molecules in B cells,4 enhances expression of
CD23,5 and up-regulates the expression of the IL-4
receptor.6 IL-4 also has an important role in tissue
adhesion.7
2. Results and discussion
A ceramide library was synthesized by a solid phase
combinatorial chemistry as previously described (Fig.
1A).11 A library of 80 compounds was generated using
8 sphingosine-like core structures and 10 acyl groups
(Fig. 1B). The synthetic compounds were characterized
1
by high-resolution mass spectrometry and H NMR to
confirm their identity and purity.
To examine the effects of the ceramide compounds on
IL-4 production, EL4 T cells were stimulated in vitro
for 48 h with PMA in the absence or presence of cera-
mide derivatives. As indicated in Table 1 and Figure 2,
Keywords: Ceramide library; Interleukin-4; T cells; Allergy.
*
Corresponding author. Tel.: +8262530 9235; fax: +8262530
2911; e-mail: taekim@chonnam.ac.kr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.027

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J. Park et al. / Bioorg. Med. Chem. 13 (2005) 2589–2595
Figure 1. Synthesis and structures of ceramides. Full names for the abbreviations used for compounds; [Cores] DES: D-erythro-sphingosine, DTS: D-
threo-sphingosine, LTS: L-threo-sphingosine, 10ES: 10-carbon-sphingosine, 20ES: 20-carbon-sphingosine, 15ES: 15-carbon-sphingosine, PC:
psychosine, AP: 2-(aminomethyl)pyridine; [Tails] 1: acetyl, 2: propionyl, 3: butyryl, 4: valeryl, 5: hexanoyl, 6: benzoyl, 7: decanoyl, 8: lauroyl, 9:
myristoyl, 10: palmitoyl.
many of ceramide derivatives showed inhibitory activi-
ties on IL-4 production by PMA-stimulated EL4 cells.
Especially, four ceramide derivatives with a lauroyl
group, A8, E8, G8 and H8, were more potent than other
effective ceramide derivatives in inhibiting IL-4
production.
ing amounts of A8, E8, G8 and H8 (1, 5, 10 lM) for
48 h and the levels of IL-4 were determined by IL-4 ELI-
SA. As shown in Figure 3A, each of four ceramide
derivatives significantly inhibited IL-4 production from
PMA-stimulated EL4 T cells in a dose-dependent man-
ner. To examine whether the IL-4 production inhibited
by these ceramide derivatives at the protein level corre-
lates with mRNA transcription, IL-4 mRNA levels were
analyzed in PMA-stimulated EL4 cells in the absence or
To confirm these inhibitory effects, EL4 T cells were
stimulated with PMA (1 ng/mL) in the presence of vary-

J. Park et al. / Bioorg. Med. Chem. 13 (2005) 2589–2595
Table 1. Inhibition of a ceramide library on IL-4 production
2591
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
48.83 10.98
88.29 4.28
84.01 9.36
77.22 15.95
79.84 3.64
89.49 5.28
74.69 11.81
34.25 4.93
66.59 8.31
68.00 1.82C10
78.57 7.03
77.76 3.74
77.96 6.24
83.06 2.97
111.56 2.41
130.42 3.91
74.44 10.58
63.48 4.43
69.06 1.95
72.47 3.44
C1
C2
C3
C4
C5
C6
C7
C8
C9
84.66 3.37
96.52 6.03
113.36 5.93
114.24 7.96
146.23 2.67
111.28 2.41
92.00 9.08
70.49 5.83
79.10 3.95
5.690.72
E1
E2
E3
E4
E5
E6
E7
E8
E9
78.58 7.09
84.77 4.02
99.77 3.36
87.25 5.26
96.92 7.44
90.77 1.75
80.89 10.52
50.33 2.77
85.39 8.96
G1
G2
G3
G4
G5
G6
G7
G8
G9
85.64 11.03
88.26 2.48
111.14 4.62
112.84 13.84
92.17 4.51
89.16 3.88
51.46 3.86
47.16 4.51
80.18 4.22
.728E0.1805
2
G10
081.76 5.2
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
86.25 7.37
90.39 4.77
83.35 10.41
54.15 7.22
86.64 5.26
76.71 4.37
96.30 15.15
73.15 0.80
78.09 4.85
77.12 3.45
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
91.31 12.21
89.51 3.72
96.51 6.87
95.54 954
89.98 5.68
93.57 1.75
78.67 12.47
74.96 9.16
86.18 6.69
80.46 7.24
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
93.99 7.41
97.28 2.99
103.77 7.66
96.08 8.62
90.17 2.91
90.73 5.28
94.20 12.11
48.87 2.62
95.13 8.44
107.33 0.47
EL4 T cells were stimulated with PMA in the absence or presence of 10 lM of each ceramide derivatives for 48 h and the IL-4 levels in the culture
supernatants were determined by IL-4-specific ELISA. The data are represented as % inhibition relative to the IL-4 level of PMA-stimulated EL4
cells in the absence of ceramide compounds. The values are the mean standard deviations of triplicate determinations.
with KLH in the absence or presence of A8, E8, G8
and H8. As shown in Figure 4A, four derivatives signif-
icantly inhibited IL-4 production in KLH-primed lymph
node cells. A8, E8, G8 and H8 also inhibited IL-4
mRNA levels in KLH-primed lymph node cells, clearly
confirming that the changes in IL-4 production with A8,
E8, G8 and H8 occurred at the transcriptional level (Fig.
4B). Importantly, ceramide derivatives did not affect the
mRNA levels of interferon-c, a Th1 cytokine (Fig.
4C), suggesting that the inhibitory effect of ceramide
derivatives might be specific for IL-4. IL-4 inhibition
by ceramide derivatives did not result from a general
cytotoxic effect since cellsÕ viability in all cultures re-
mained constant throughout the incubation period in
the presence of ceramide derivatives used in the experi-
ment, as demonstrated by trypan blue exclusion test
(data not shown).
Figure 2. The inhibitory effect of ceramide derivatives on IL-4
production. The Z axis is % inhibition relative to the IL-4 level of
PMA-stimulated EL4 T cells in the absence of ceramide compounds.
IL-4, mainly produced in CD4+ Th2cells, is an impor-
tant stimulus for the switching of antibody isotype to
IgE in both mice and humans.12 Higher than normal
serum IgE is often found in patients with allergic dis-
presence of ceramide derivatives with a lauroyl group.
As shown in Figure 3B, A8, E8, G8 and H8 inhibited
IL-4 mRNA levels in PMA-activated EL4 T cells, indi-
cating that the changes in IL-4 production with A8, E8,
G8 and H8 occurred at the transcriptional level. Treat-
ment with A8, D8, E8 and H8 did not affect b-actin
mRNA expression by PMA-activated EL4 T cells, sug-
gesting that the inhibitory effect of IL-4 production by
A8, E8, G8 and H8 was not the result of a generalized
inhibition of cellular activation.
eases, including allergic asthma. Reduction of IgE is
considered as one strategy in the treatment of asthma.13
In this study, the inhibition of IL-4 production by A8,
E8, G8 and H8-treated EL4 cells and primary lymph
node cells might result in an inhibition of the IgE level
in sera, leading to the reduction of the allergic response.
Suplatast tosilate (IPD; Taiho; Tokyo, Japan) is an
immunoregulator that suppresses IgE production and
eosinophil infiltration through selective inhibition of
IL-4 and IL-5 synthesis by Th2-like cells but not IFN-
c production in Th1 cells.14
To investigate whether the inhibitory effects of four cer-
amide derivatives on IL-4 production also occurred in
primary lymph node cells, BALB/c mice were immu-
nized with KLH (100 lg) in alum. Seven days later,
the draining lymph node cells were stimulated in vitro
The reason why ceramide derivatives with a lauroyl
group were more effective than other ceramide deriva-
tives is not clear. It seems that ceramide compounds
with a lauroyl group are beneficial for the higher

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J. Park et al. / Bioorg. Med. Chem. 13 (2005) 2589–2595
Figure 3. Effects of A8, E8, G8 and H8 on IL-4 production and mRNA expression level in PMA-activated EL4 T cells. (A) EL4 T cells were
stimulated with PMA (1 ng/mL) in the absence or presence of varying amounts of A8, E8, G8 and H8 (1, 5, 10 lM) for 48 h. The cell culture
supernatants were harvested and assayed for IL-4 levels by ELISA. The values represent the means standard deviations of triplicate determinations
from one representative experiment. The experiment was repeated more than three times with similar results. *P < 0.0001 and **P < 0.00005,
compared with the respective PMA-treated cells. (B) EL4 cells were stimulated with PMA (1 ng/mL) in the absence or presence of A8, E8, G8 and H8
(1, 10 lM) for 6 h. Cellular RNA from each treatment was extracted and the mRNA expression for IL-4 and b-actin was analyzed by RT-PCR.
potency in inhibiting IL-4 production at the transcrip-
tional level. Several transcription factors have been iden-
tified that are critical for IL-4 transcription, including
Stat6,15 NF-ATc,16 c-Maf,17 GATA-318 and JunB.19
To date, direct binding to and/or activation of the IL-
4 promoter has been demonstrated for NF-ATc,20 c-
Maf17 and JunB,19 but not for Stat621 or GATA-3.22
The transcription factor NF-AT is well known to play
an essential role in the inducible transcription of IL-4
gene during T cell activation since human and murine
IL-4 gene promoters contain at least four NF-AT sites
that control their induction in T cells.23 Activator pro-
tein (AP)-1 is necessary for the full high-levels of IL-4
production in atopic Th2cells and is also an important
contributor to the expression of Th2cytokines, IL-5 and
IL-13 as well as IL-4.24 IL-4 gene promoter contains at
least two AP-1 sites, P1 and P4.25 Additionally, cera-
mide has been shown to activate a diverse set of protein
kinases and protein phosphatases.26 Therefore, A8, E8,
G8 and H8 may regulate IL-4 gene transcription by
inhibition activities of transcription factors, such as
NF-AT or AP-1, through the modulation of diversity
signalling molecules. The structure–reactivity relation-
ships for the inhibitory activity of ceramide are being
elucidated.
In conclusion, we have demonstrated that some cera-
mide derivatives, A8, E8, G8 and H8, strongly inhibited
IL-4 production in both EL4 T cells and primary lymph
node cells. This effect may explain the anti-allergic
effects of A8, E8, G8 and H8. Because the ratio of
IFN-c-secreting Th1 and IL-4-secreting Th2cells is
closely correlated with the outcome of many diseases,27
A8, E8, G8 and H8 may protect patients from develop-
ing diseases caused by unwanted Th2-dominated
responses.

J. Park et al. / Bioorg. Med. Chem. 13 (2005) 2589–2595
2593
Figure 4. Effects of A8, E8, G8 and H8 on IL-4 production and mRNA expression in KLH-primed lymph node cells. (A) Mice were injected into the
footpad with KLH (100 lg) in alum. Seven days later, the lymph node cells were collected and stimulated in vitro for 4 days with KLH (100 lg/mL)
in the absence or presence of varying amounts of A8, E8, G8 and H8 (1, 10 lM). *P < 0.005 and **P < 0.0005, compared with the PMA-treated cells
in the absence of ceramide compounds. (B and C) Lymph node cells from KLH-primed mice were re-stimulated for 6 h with KLH (100 lg) in the
absence or presence of varying amounts of A8, E8, G8 and H8 (1, 10 lM). Cellular RNA from each treatment was extracted and the mRNA
expression for IL-4, IFN-c and b-actin was analyzed by RT-PCR.
3. Experimental
3.1. General synthetic methods
and washed with N,N0-dimethylformamide (3 ·
20 mL), methanol–dichloromethane (1:1, 5 · 20 mL)
and dichloromethane (5 · 20 mL) and dried.
All chemicals were purchased from Sigma–Aldrich or
Acros, and were used without further purification. Prod-
ucts obtained as solids or high boiling-point oils were
dried under vacuum. Analytical thin-layer chromatogra-
phy was performed on precoated silica plates (Merck 60-
F 254, 0.25 mm thickness); compounds were visualized
by staining with 10% phosphomolybdic acid in ethanol
solution or 10% ninhydrin in ethanol solution.
3.3. General procedure for ceramide synthesis
An activated ester resin (50 mg, 75 lmol) was added to a
core amine (3 lmol) in THF (1 mL) and agitated on a
shaker overnight at room temperature. The reaction
mixture was filtered and washed with a portion of
THF (1 mL) and the filtrate was combined and dried.
Completion of the reaction was confirmed by TLC
and negative ninhydrin staining. The purity of each
compound was checked by TLC and phosphomolybdic
acid staining; each gave a single spot.
3.2. General procedure for activated ester synthesis using
acyl chloride
Activated ester resins were prepared by following litera-
ture procedure with a slight modification.12a Nitrophe-
nol resin (1 g, 1.73 mmol) was suspended in 1-methyl-
2-pyrrolidinone (10 mL). After 10 min, pyridine (1 mL,
12.4 mmol) and the appropriated acyl chloride (4 mmol,
except the resin 1, which was prepared by acetic anhy-
dride) were added to the reaction mixture and stirred
overnight at room temperature. The resin was filtered
3.4. Materials, cell culture and mice
Phorbol 12-myristate 13-acetate (PMA) was purchased
from Sigma (St. Louis, MO). KLH was from Calbio-
chem Co. (San Diego, CA). Anti-murine IL-4 mAb
11B11 and BVD6 were from M. Howard, DNAX Re-
search Institute (Palo Alto, CA) and recombinant mur-
ine IL-4 were purchased from PharMingen (San Diego,

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J. Park et al. / Bioorg. Med. Chem. 13 (2005) 2589–2595
CA). Cultures of EL4 thymoma cells and lymph node
cells were maintained in RPMI-1640 medium (Gibco
BRL, Grand Island, NY) supplemented with 10% fetal
bovine serum (FBS; Hyclone Laboratories, Logan,
UT) and 1% penicillin/streptomycin at 37 ꢁC in a 5%
CO2 humidified air atmosphere. Six to eight-week-old-
female BALB/c mice were obtained from Daehan Ani-
mal Inc. (Seoul, Korea), and maintained in pathogen-
limited conditions. The mice were treated according to
the National Institutes of Health Guidelines for the Care
and Use of Laboratory Animals.
AAC GCA GCT CAG TAA CAG TCC G-30). The
PCR reactions were run for 35 cycles for 94 ꢁC (30 s),
57 ꢁC (30 s), 72 ꢁC (30 s) using a PCR Thermal Cycler
(MJ Research, Watertown, MA). After the amplifica-
tion, the RT-PCR products were separated in 1.5% (w/
v) agarose gels and stained with EtBr. The sizes of
PCR products for IL-4 and b-actin genes were 397 bp
and 349 bp, respectively.
3.8. Statistical analysis
StudentÕs t-test was used to determine the statistical dif-
ferences between various experimental and control
groups. P-values <0.05 were considered significant.
3.5. In vitro stimulation of lymph node cells
Draining axillary, popliteal and inguinal lymph nodes
were removed from mice 7 days after priming with
100 lg KLH absorbed to aluminium hydroxide (alum)
adjuvant in the foot-pads as previously described.28 Sin-
gle-cell suspensions of lymph nodes were prepared and
cultured in vitro with KLH (100 lg/mL) in the absence
or presence of either ceramides. At the indicated times
as described in the figure legends, the levels of IL-4 in
the cell supernatants were determined by sandwich en-
zyme-linked immunosorbent assay (ELISA), and
mRNA levels of IL-4 and interferon-gamma (IFN-c)
in the cells were assayed by reverse transcription-poly-
merase chain reaction (RT-PCR).
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