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IMMUNOBIOLOGY
From the Departments of Medicine and Otolaryngology,
The Johns Hopkins School of Medicine, Baltimore, MD.
Previous studies indicated that aspirin (acetylsalicylic acid
[ASA]) can have profound immunomodulatory effects by regulating cytokine gene expression in several types of cells. This study is the
first in which concentrations of ASA in the therapeutic range were
found to significantly reduce interleukin (IL)-4 secretion and RNA
expression in freshly isolated and mitogen-primed human CD4+ T cells. In contrast, ASA did not affect IL-13,
interferon- Aspirin (acetylsalicylic acid [ASA]) is the
oldest and most widely used nonsteroidal anti-inflammatory drug
(NSAID). Although several other classes of NSAIDs have become available
since the introduction of ASA in 1899, this agent and structurally
related salicylates still provide the mainstay of therapy for
inflammatory musculoskeletal disorders. In addition, these compounds
have been shown to be effective in the management and prevention of an
increasingly diverse array of noninflammatory conditions, including
coronary and cerebral ischemia and gastrointestinal
cancer.1,2
Both the therapeutic properties of NSAIDs and their side effects have
been ascribed to their ability to inhibit generation of prostaglandin
(PG) and thromboxane by interfering with the intracellular enzyme
cyclo-oxygenase (COX).3 It is widely accepted that the
anti-inflammatory actions of NSAIDs are mediated by inhibition of the
inducible COX isoform COX-2, whereas their detrimental effects on
gastric mucosa viability and platelet function are due mostly to
inhibition of COX-1.4 The relative effectiveness of
several NSAIDs against the 2 isozymes varies
considerably.5 In particular, although ASA is a relatively
effective, irreversible inhibitor of COX-1, its effects on COX-2
activity are negligible.5 This probably explains why
higher doses of ASA are required in the treatment of chronic
inflammatory diseases than are sufficient to inhibit PG generation in
different experimental models in vitro.6 However, the in
vivo anti-inflammatory and anticancer activity of nonacetylated
salicylates, which are poor overall inhibitors of both COX-1 and COX-2,
is almost superimposable to that of ASA or even more potent NSAIDs,
such as diclofenac.7 Indeed, given the short serum
half-life of ASA (15 minutes), the serum concentrations of salicylic
acid (SA), its major nonacetylated metabolite, are better predictors of
therapeutic effectiveness than the concentrations of ASA
itself.6
In the light of these observations, it has been speculated that
inhibition of PG production cannot fully account for the therapeutic potential of ASA and related salicylates.6 Indeed, several studies showed that these compounds have a spectrum of biochemical and
pharmacologic effects that are not related to COX inhibition and not
shared with other NSAIDs.8-15 A major finding was the discovery that ASA and SA can interfere with the activation of critical
transcription factors, such as nuclear factor (NF) It has long been speculated that the effectiveness of ASA in chronic
inflammation, as well as in several apparently unrelated clinical
conditions, might be at least partly accounted for by its effects on
immune responses.18 However, data on this issue are sparse
and contradictory. In particular, few studies have assessed the effects
of ASA or other NSAIDs on T-cell differentiation and function.
Decreased production of immunomodulating PGs, such as prostaglandin
E2 (PGE2), in accessory cells may account for the ability of ASA and other NSAIDs to bolster T helper (Th)1-driven cellular immune responses.19,20 ASA and ibuprofen can
enhance mitogen-induced T-cell proliferation and the expression of
interleukin-2 (IL-2), IFN- In this study, we examined the effect of ASA on the expression of
effector cytokines in purified human CD4+ T cells. The
study is the first to find that therapeutic concentrations of ASA can
significantly and selectively inhibit the expression of the
Th2-associated cytokine IL-4. This effect of ASA is not associated with
reduced cell viability or detectable apoptotic changes and is
apparently not due to inhibition or acetylation of COX isozymes in T
cells, since it is not shared with other NSAIDs and can be reproduced
in experiments using identical concentrations of the nonacetylated
salicylate SA. Our findings in purified CD4+ T cells
provide the first evidence that these cells may be a direct target of
ASA and related compounds. We found that inhibition of IL-4 expression
by ASA and SA occurs at the transcriptional level and is due to
interference with the binding of a Ca++-inducible factor to
a proximal IL-4 promoter element. Much evidence indicates that this
factor is not NF- Cell isolation and culture
Cell stimulation and cytokine detection
RNA isolation and analysis The expression of cytokine transcripts was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA extracted 6 hours after stimulation with the Trizol reagent (Life Technologies).31 The primers used were 5'-TCCCAACTGCTTCCCCCTCTG-3' (forward IL-4), 5'-TGCTTGTGCCTGTGGAACTGC-3' (reverse IL-4), 5'-AAGGCTCCGCTCTGCAATGG-3' (forward IL-13), 5'-GGGCCACCTCGATTTTGGTGT-3' (reverse IL-13), 5'-GCATCCAAAAGAGTGTGGAGACCATC-3' (forward IFN- ),
5'-CGACCTCGAAACAGCATCTGACT-3' (reverse IFN-),
5'-CATGCCCAAGAAGGCCACAGA-3' (forward IL-2), and 5'-GCTGTCTCATCAGCATATTCACACATGA-3' (reverse IL-2) (all from Genosys, The Woodlands, TX).
Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from
Stratagene (La Jolla, CA). IL-4 transcripts were quantified by
real-time RT-PCR.32 Total RNA (10 ng/sample) was subjected to RT-PCR using the GeneAmp Gold kit (Applied Biosystems, Foster City,
CA). Quantitative determination of the amplified products was done with
an ABI Prism 7700 System (Applied Biosystems). The forward and reverse
primers were 5'-CGACTGCACAGCAGTTCCA-3' and 5'-CAGGCCCCAGAGGTTCC-3',
respectively (Applied Biosystems). The detection probe, labeled with
the reporter dye 6-carboxy-fluorescein ( Transient transfections and analysis of reporter gene expression The IL-4.265, IL-4.225, IL-4.145, IL-4.95, and IL-4.65 chloramphenicol acetyltransferase (CAT) plasmids were constructed by insertion in the HindIII and XbaI sites of pCAT-Basic (Promega, Madison, WI) of PCR-generated fragments spanning base pairs (bp) 265, 225, 145, 95, and 65, respectively, to
+55 of the human IL-4 gene.30,33 The IL-2.15 CX CAT
plasmid (IL-2.312), including bp 312 to +55 of the human IL-2 gene,
was donated by Dr Gerald R. Crabtree (Stanford University, Stanford,
CA).34 Plasmids (1 µg) were transfected into
106 Jurkat cells by 48-hour incubation in 3 mL complete
medium containing 5 µg/mL SuperFect (Qiagen, Valencia,
CA).35 Cells were treated as indicated 20 hours before
harvest. Expression of CAT was measured by using a commercial ELISA
(Roche, Indianapolis, IN) and normalized by considering the
total protein in each sample (Bio-Rad, Hercules, CA).
Electrophoretic mobility shift assays Jurkat cells (2.5 × 107/condition) were lysed in 10 mM HEPES (pH 7.9), 30 mM potassium chloride (KCl), 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 µg/mL leupeptin, 1 µg/mL aprotinin, and 0.075% Nonidet P40 (Sigma). Nuclei were extracted in 20 mM HEPES (pH 7.9), 420 mM KCl, 1 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 µg/mL leupeptin, 1 µg/mL aprotinin, and 20% glycerol. Protein concentrations were measured by using the Bradford reagent (Bio-Rad). The following oligonucleotides spanning IL-4 promoter elements (Figure 5B) were synthesized (Genosys): 5'-TTACCTGTTTGTGAGGCATTTTTTC-3' (IL-4.155), 5'-TTTTCTCCTGGAAGAGAGGTGCTGA-3' (IL-4.135), 5'-TTTTCTCCTGGAAGAGAGGTGCTGA-3' (IL-4.115), and 5'-GAGAGGTGCTGATTGGCCCCAAGT-3' (IL-4.CCAAT).These oligonucleotides, as well as a 22 mer spanning a consensus
NF-
Aspirin selectively inhibits IL-4 secretion from CD4+ T cells We studied the effects of ASA on the secretion of effector cytokines from freshly isolated CD4+ PBT. Suppression of PG production in monocytes may contribute to the reported enhancing effects of NSAIDs, including ASA, on T-cell proliferation and IFN-
and IL-2 expression.22 Although both COX-1 and COX-2 have
been reported to be expressed in human T cells, the importance of this
finding is not clear.36 To minimize the contribution of
monocytes or other accessory cells to the possible effects of ASA on
T-cell activation, we purified CD3+CD4+ cells
to at least 97% by means of negative selection from enriched PBT preparations.
CD4+ PBT were treated with increasing concentrations of ASA
(10
Similar results were obtained with enriched preparations containing
about 45% CD4+ PBT and substantial percentages of
accessory cells. Figure 1C shows the effect of 10 COX inhibition and acetylation play no role in IL-4 inhibition by aspirin To assess whether inhibition of PG generation might account for IL-4 inhibition by ASA, we compared the effects of different COX inhibitors on expression of this cytokine in purified CD4+ PBT. Figure 2 shows that among several NSAIDs, only SA, the weakest inhibitor of COX-1 and COX-2 in this pharmacologic group,5 inhibited IL-4 expression to an extent comparable to that of ASA. In striking contrast to this finding, the potent COX inhibitors FBP and IM, used at concentrations in or above their respective COX-inhibitory and therapeutic ranges,5 did not affect IL-4 expression (Figure 2). Neither of these compounds significantly affected IL-2 secretion.
In these experiments, ASA or other NSAIDs did not affect cell
viability, as assessed by trypan blue or propidium iodide exclusion. Even longer incubations (up to 72 hours) of enriched or purified CD4+ PBT with 10 Aspirin inhibits IL-4 gene expression at the transcriptional level Decreased IL-4 secretion was paralleled by reduced accumulation of IL-4 message in CD4+ PBT treated with ASA. Figure 3A shows the inhibitory effect of 10 3 M ASA on IL-4 RNA expression in cells stimulated with
0.5 µg/mL A23187 and 10 ng/mL PMA. Also shown is that this
concentration of ASA did not affect IL-13, IFN-, or IL-2 expression
in these experiments. The degree of IL-4 RNA inhibition by ASA was
analyzed by using real-time quantitative RT-PCR. In 3 such experiments, IL-4 RNA levels were decreased by 56.4% ± 7.3% and 71.2% ± 6% in
CD4+ PBT treated with 103 M and
3 × 103 M ASA, respectively (Figure 3B). A comparable
decrease was observed in cells treated with the same concentrations of
SA (data not shown), whereas 105 M IM had no effect
(Figure 3B). Similar results were obtained in the Jurkat human T-cell
line (data not shown).
To investigate the molecular mechanisms mediating the inhibitory effect
of salicylates on IL-4 gene expression, we analyzed the effects of
these compounds on IL-4 promoter activity in transiently transfected
Jurkat cells. As shown in Figure 3C, ASA
(10 IL-4 gene inhibition by aspirin is not associated with reduced
NF- 3 M ASA. A
concentration 2.5 to 5 times higher was previously reported to be
necessary for comparable inhibition of NF- B DNA-binding and
transcriptional activities.8,26 To determine whether lower concentrations of salicylates might affect NF-B activation under our
experimental conditions, we conducted electrophoretic mobility shift
assays (EMSAs) using nuclear extracts from Jurkat cells treated with
ASA or the related compound SSA, a known potent and selective inhibitor
of NF-B.40 Figure 4A shows an experiment in
which stimulation with A23187 (0.5 µg/mL) and PMA (10 ng/mL) resulted
in formation of 2 complexes on an oligonucleotide spanning a consensus
NF-B element from the -light-chain gene (lane 2). Consistent with
previous findings,41 neither complex formed when extracts
from cells stimulated with A23187 alone were used (lane 5). Antibodies
raised against the p65 (lane 3) and p50 (lane 4) NF-B subunits
interfered with formation of both complexes. Treatment with
concentrations of ASA that significantly inhibited IL-4 expression
(Figure 4A shows the effect of 2 × 103 M) did not
affect formation of either complex (lane 6). In contrast, a similar
concentration of SSA (2 × 103 M) was, as reported
previously,40 sufficient to repress NF-B binding in
Jurkat cells completely (lane 7).
The effect of SSA on NF- Identification of a salicylate-targeted region in the human IL-4 promoter To map the promoter element mediating IL-4 gene inhibition by ASA, we generated a panel of IL-4 promoter deletions for use in reporter studies. ASA and SA (103 M) were equally effective at
inhibiting the activation of IL-4 promoter constructs truncated at bp
265 through 145 (data not shown). Figure
5A shows that CAT expression was
significantly inhibited in Jurkat cells transfected with a construct
carrying an IL-4 promoter fragment spanning bp 145 to +55 (IL-4.145)
and treated with ASA or SA (103 M) but not FBP
(105 M). The same concentrations of ASA and FBP but not
SA up-regulated the transcriptional activity of an IL-4 promoter
construct (IL-4.95) lacking bp 145 to 96 (Figure 5A). This
indicated that an element mediating inhibition of IL-4 transcription by
ASA and SA is located between bp 145 and 96 of the human IL-4
promoter and that a COX-related mechanism might account for increased
transcriptional activity of the IL-4.95 construct in cells treated with
ASA or FBP.
The sequence of the salicylate-targeted region of the human IL-4
promoter is shown in Figure 5B. Integrity of this region appears to be
critical for maximal IL-4 promoter activity in murine and human T
cells.43,44 An inverted CCAAT box at bp To understand the relative contribution of each of these elements and
their cognate factors to IL-4 transcriptional regulation by
salicylates, we generated 3 oligonucleotides spanning overlapping sequences in a region including bp
This study is the first to find that therapeutic concentrations of
ASA37 significantly inhibit IL-4 gene expression in
activated CD4+ T cells. Inhibition of IL-4 secretion in
cells treated with ASA was not associated with reduced viability,
impaired basic biochemical and molecular functions, or expression of
early apoptotic markers. Indeed, depending on the stimulants used, IL-2
production was not inhibited or was moderately enhanced after treatment
with ASA. Similarly, ASA did not affect expression of the effector cytokines IL-13 and IFN- To our knowledge, our experiments using purified PBT and Jurkat cells
provide the first evidence that CD4+ T cells may be a
direct target of ASA and related compounds. The hypothesis that
inhibition of immunomodulatory PGs mediates the effect of ASA on IL-4
production in these cells19,20 was not validated by our
experiments using structurally unrelated COX inhibitors (Figures 2 and
5A). Moreover, T cells have been reported to produce negligible amounts
of known COX products, with almost 100-fold-lower PGE2
levels than required to modulate cytokine
production.21,24,36 Previous studies using peripheral blood mononuclear cell or unfractionated PBT preparations showed that
submillimolar concentrations of ASA can enhance several aspects of
mitogen-induced T-cell activation, including proliferative responses
and the expression of IFN- None of the concentrations of ASA used in our study significantly
affected production of IL-13 and IFN- ASA inhibits NF- ASA effectively inhibited IL-4 promoter activation in cells stimulated
with a Ca++ ionophore (Figure 3B). Although the engagement
of Ca++-delivered signals is necessary and sufficient for
maximal activation of the IL-4 gene in several T-cell lines and clones,
including Jurkat cells,30,50,51 our study confirmed that
PKC coactivation by PMA is a stringent requirement for NF- The mechanisms regulating IL-4 gene expression in human and murine
CD4+ T cells have been the focus of intensive investigation
during the past few years.53 Members of the NFAT family of
transcription factors are thought to have a critical role in the
activation of antigen-dependent IL-4 gene expression in T
cells.50,54 Although the known NFAT molecular species
appear to be expressed at similar levels in Th1 and Th2 cells,
NFAT-directed IL-4 transcription is preferentially induced in Th2
cells, presumably because of the involvement of lineage-restricted
coactivators.55 NFAT, however, is not a reasonable
candidate for a molecular target of salicylates in our experimental
system. Ca++- and calcineurin-dependent activation of NFAT
also appears to be a requirement for expression of IFN- Taken together, our observations are consistent with the idea that a
previously unrecognized transcriptional target accounts for the
inhibitory effect of salicylates on IL-4 expression. In vitro and in
vivo evidence suggests that the proximal 88 bp of the IL-4 promoter,
including the NFAT-binding P0 and P1 elements (Figure 5B), are
sufficient to mediate proper lineage-restricted and mitogen- or
antigen-induced IL-4 expression in distinct Th subsets.55,61 However, our finding that IL-4 gene
inhibition by salicylates involves DNA-protein interactions in a region
upstream of bp IL-4, the prototypic cytokine expressed in Th2 cells, plays a pivotal
role in the regulation of hematopoiesis and immune and inflammatory
responses and is involved in the pathogenesis of a wide spectrum of
disease conditions.62-65 Although suppression of Th1
responses by means of NF-
We thank Drs Gerald R. Crabtree and Jack L. Strominger for the generous gift of reagents; Drs Susan M. MacDonald, Robert P. Schleimer, and Bradley J. Undem for critical reading of the manuscript; and Drs Bruce S. Bochner, Katsushi Miura, and Cristiana Stellato for helpful discussions and comments.
Submitted March 6, 2000; accepted October 27, 2000.
Supported by grants AI41463 (to V.C.), AI07290 (to L.M.L.), and AI01152 (to S.N.G.) from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Vincenzo Casolaro, The Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224; e-mail: casolaro{at}mail.jhmi.edu.
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M. Aceves, A. Duenas, C. Gomez, E. San Vicente, M. S. Crespo, and C. Garcia-Rodriguez A New Pharmacological Effect of Salicylates: Inhibition of NFAT-Dependent Transcription J. Immunol., November 1, 2004; 173(9): 5721 - 5729. [Abstract] [Full Text] [PDF]
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M. Perez-G., M. Melo, A. D. Keegan, and J. Zamorano Aspirin and Salicylates Inhibit the IL-4- and IL-13-Induced Activation of STAT6 J. Immunol., February 1, 2002; 168(3): 1428 - 1434. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, tumor necrosis factor-
, and IFN-
max, 518 nm) at
the 5' end and the quencher dye 6-carboxytetramethyl-rhodamine (
97% CD3+CD4+) treated with a therapeutic
concentration of ASA (10
, 595.1 ± 28.9 pg/mL) and IL-2 (
, 364.4 ± 39.7 U/mL)
secretion in 4 independent experiments done in duplicate is shown. The
asterisk indicates P < .05 (Wilcoxon test) relative to
DMSO-treated controls. (C) IL-4 concentrations in the supernatants of
enriched PBT preparations (33%-54% CD3+CD4+)
treated with 10
) or 0.1% DMSO (
) before
stimulation with anti-CD3 ([
in particular, the frequencies of IL-4-expressing
cells compared with IFN-
,
10
,
10
, 10