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Centro de Biología Molecular, "Severo Ochoa," Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-
production
and cell proliferation, but not the expression of CD69, an immediate
early gene. COX-2 inhibitors also abolished induced transcription of
reporter genes driven by IL-2 and TNF- promoters. Moreover, induced
transcription from NF-
B- and NF-AT-dependent enhancers was also
inhibited. These results may have important implications in
anti-inflammatory therapy and open a new field on COX-2-selective
nonsteroidal anti-inflammatory drugs as modulators of the immune
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-, and GM-CSF. The
coordinated production of these cytokines is crucial for the regulation
of an immune response because they control the proliferation,
differentiation, and function of lymphoid and nonlymphoid cells. On the
other hand, activated T cells play an important role in the
pathogenesis of inflammatory diseases, initiating and sustaining
inflammation (4, 5). Proinflammatory cytokines secreted by T cells
activate the inflammatory activity of macrophages and granulocytes. As
a consequence, a new wave of inflammatory mediators, including
cytokines such as TNF-, IL-1ß, eicosanoids, reactive oxygen
intermediates, and nitric oxide, is produced that further augment
inflammation (6). Eicosanoids, including PGs, thromboxanes, and leukotrienes, are metabolites of the unsaturated fatty acid arachidonic acid (AA),3 synthesized and secreted by most human tissues and cell types (7, 8). Among the cells of the immune system, it is generally accepted that AA metabolites are mainly produced by accessory cells (9, 10). On the other hand, it is well known that eicosanoids play a key role in the regulation of both humoral and cell-mediated immunity, modulating cytokine and Ig production as well as T cell proliferation and activation (11, 12, 13, 14).
Prostaglandin H endoperoxide synthase or cyclooxygenase (COX) catalyzes
the two-step conversion of AA to PGH2, the first
reaction required for the biosynthesis of PGs and thromboxanes. At
least two isoforms of the enzyme are expressed in mammalian tissues,
COX-1 and COX-2. COX-1 is constitutively expressed in most mammalian
tissues and is thought to be involved in homeostatic prostanoid
biosynthesis (15, 16). In contrast, COX-2 is induced by various
proinflammatory agents, including cytokines and mitogens (15, 17).
COX-2 is thought to be the predominant isoform involved in the
inflammatory response. Accordingly, the ability of nonsteroidal
anti-inflammatory drugs (NSAIDs) to inhibit COX-2 activity may
explain their therapeutic effects as anti-inflammatory drugs,
whereas inhibition of COX-1 activity may account for some of their
unwanted side effects (16, 18). Therefore, most of the new research on
anti-inflammatory drugs has been aimed at targeting the
COX-2-inducible production of PGs. Newly developed drugs that have high
selectivity against COX-2, such as NS398, Celecoxib, or Meloxicam, have
been proved to be potent anti-inflammatory compounds without
causing gastric toxicity (19, 20, 21). However, there is growing evidence
that some NSAIDs may have additional immunomodulatory properties not
apparently related to the inhibition of PG synthesis. Thus, salicylates
and aspirin, besides inhibiting COX-1, have also been described as
inhibitors of the activation of the NF-B as well as
NF-B-dependent gene expression (22). Tenidap and Tepoxalin, both
cyclooxygenase and lipoxygenase inhibitors, suppress proliferation and
IL-2 or IFN- expression in activated T cells (23, 24, 25). Besides,
several NSAIDs, including Indomethacin, Ibuprofen, and Fenoprofen, have
been recently shown to act as agonists of the transcription factor,
peroxisome proliferator-activated receptor (PPAR)-, inhibiting
PMA-induced cytokine synthesis in human peripheral blood monocytes
(26, 27).
All of these observations led us to investigate the expression,
regulation, and functional role of COX-1 and COX-2 during human T cell
activation. Our results clearly show that upon TCR/CD3 triggering,
COX-2 mRNA and protein expression were rapidly induced in T cells.
COX-2-selective inhibitors such as NS398 and Celecoxib, but not
Indomethacin, a nonselective NSAID, negatively regulated proliferation,
cell surface expression of activation markers, and cytokine production
by activated T cells. These effects could be attributable to
down-regulation of the transcription of these genes through inhibition
of transcription factors such as NF-B and NF-AT. Thus, the results
presented in this study provide the first evidence that COX-2 is
transcriptionally up-regulated in T cells and that it behaves as early
inducible gene involved in the T cell activation process. Our results
suggest that COX-2-selective NSAIDs are immunosuppressive drugs and
could have important applications in anti-inflammatory therapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human Jurkat T leukemia cell line was grown in complete RPMI 1640 medium supplemented with 10% FCS. Purified human T lymphocytes were obtained from buffy coats of healthy donors by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) centrifugation (28). The PBL fraction was plated and adherent cells were removed. Purified T cells were obtained by passing the nonadherent population through a nylon fiber wool column, as previously described (28). The purity of the population, detected by flow cytometry, was always greater than 95% CD3+ cells.
Activation of human T lymphocytes through the TCR/CD3 complex and the CD28 receptor was conducted by adding the purified T cells to plates coated with anti-CD3 Ab (2 µg/ml), followed by subsequent addition of anti-CD28 Ab (250 ng/ml) (Immunokontact, Bioggio, Switzerland). Jurkat cells were stimulated by PMA (Sigma, St. Louis, MO), A23187 ionophore (Sigma), or immobilized anti-CD3 Ab (28), as indicated. Cycloheximide (CHX; 5 µg/ml) or actinomycin D (ActD; 5 µg/ml) was added to cells 45 min, and cyclosporin A (CsA; 100 ng/ml) (Sandoz, East Hanover, NJ), NS398 (10–100 µM) (Cayman Chemical, Ann Arbor, MI), Celecoxib (10–100 µM) (provided by Laboratorios Dr. Esteve, Barcelona, Spain), or Indomethacin (10–100 µM) (Sigma) 1 h prior to activation. None of the agents affect the viability of the cells at the concentrations used, as confirmed by the trypan blue dye exclusion test.
mRNA analysis
Total RNA was prepared from Jurkat or purified human T cells by the TRIzol reagent RNA protocol (Life Technologies, Paisley, U.K.). Total RNA (1 µg) was reverse transcribed into cDNA and used for PCR amplification with either human COX-1, COX-2, CD69, or GAPDH-specific primers by the RNA PCR Core Kit (Perkin-Elmer, Norwalk, CT), as previously described (29). The sequences of the sense and antisense primers for CD69 were: sense, 5'-GCACCATGTTCAGAACAAGC-3'; antisense, 5'-TGAAGGGTCCTTCCAAGTTC-3'. Briefly, the PCR was amplified by 20–35 repeat denaturation cycles at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Amplified cDNAs were separated by agarose gel electrophoresis and bands visualized by ethidium bromide staining. For quantitation of amplified cDNA, dot-blot analysis was conducted with human COX-1, COX-2, CD69, and GAPDH oligonucleotide radioactive probes complementary to internal sequences of the cDNA amplified. Radioactivity in the COX bands or dots was quantified by a phosphorimager and normalized with respect to the GAPDH values from parallel samples.
Northern blot analysis
Poly(A)+ mRNA was purified by affinity chromatography on oligo(dT) cellulose. A total of 2 µg of poly(A)+ mRNA was separated on formaldehyde agarose gels and blotted onto nylon membranes according to standard protocols (30). Human COX-1, COX-2, and GAPDH probes corresponding to the cDNA fragments amplified by RT-PCR were labeled by the random priming method (30). After hybridization and washes as previously described (31), the blots were then exposed to x-ray film for autoradiography.
Immunoblot analysis
Cells were disrupted in ice-cold lysis buffer (50 mM Tris-HCl (pH 8), 10 mM EDTA, 50 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF). Solubilized extracts (50 µg) were separated by SDS-PAGE on 10% polyacrylamide gel, and electrophoretically transferred to nitrocellulose filters. After blocking for 2 h with 5% nonfat dried milk in TBST (Tris-buffered saline containing 0.1% Tween-20), the membranes were incubated overnight at 4°C with monoclonal mouse anti-COX-2 (1:250) (Transduction Laboratories, Lexington, KY) in blocking buffer. The filters were washed and incubated for 1 h with horse anti-mouse IgG secondary Ab linked to horseradish peroxidase (Pierce, Rockford, IL) at 1/15,000 dilution, and the stained bands were visualized by the SuperSignal Substrate detection system (Pierce, Rockford, IL).
Determination of cyclooxygenase activity
Purified T lymphocytes or Jurkat cells were stimulated with anti-CD3 plus anti-CD28 Abs or PMA plus Ion, respectively. After 24-h incubation, cells were washed twice with ice-cold PBS and disrupted by sonication in ice-cold 100 mM Tris-HCl, pH 7.8. COX-2 enzymatic activity was determined with 100 µg of protein from cell sonicates in 0.2 ml vol of assay buffer (100 mM Tris-HCl (pH 7.8), 5 mM tryptophan, and 5 mM reduced glutathione). Samples were incubated at 37°C for 15 min in the presence of an excess arachidonic acid (100 µM). The reaction was stopped by boiling, and samples were centrifuged at 10,000 x g for 10 min. Concentrations of PGs were measured by a prostaglandin screen colorimetric assay kit (Cayman Chemical).
Proliferation assays
Purified T cells (1 x 105 cells/100 µl RPMI plus 2% FCS) were grown in the absence or presence of different concentrations of the indicated compounds. Cells were incubated for 36 h in a 37°C and 5% CO2 atmosphere. Cell proliferation was estimated by measuring [3H]thymidine (New England Nuclear, Boston, MA) incorporation to DNA during the last 24 h of culture. Cells were harvested through glass fiber filter paper using a multiwell cell harvester (Scatron, Lier, Norway). The amount of radioactivity incorporated was estimated in a liquid scintillation beta counter. All of the experiments were conducted in triplicate cultures.
Analysis of cell surface expression
Cell surface expression in T cell cultures was evaluated by direct flow cytometry, as previously described (28). T cells were stimulated with anti-CD3 plus anti-CD28 Abs for 24 h in the presence or absence of NSAIDs, as indicated. Surface expression of the CD69, CD25, and CD71 Ags was analyzed by flow cytometry using FITC-conjugated Abs (Becton Dickinson, San Jose, CA) in a FACScan flow cytometer (Becton Dickinson). A minimum of 5000 cells per point was analyzed. The final percentage of positive cells was obtained by subtracting the values of negative control X63-FITC-conjugated Ab from those obtained with the specific Ab.
Cytokine measurement
The concentration of IL-2, TNF-, and IFN- was quantified
using specific ELISA in supernatants of the same cell cultures used for
cell surface analysis, harvested 24 h after activation.
Commercially available kits were used according to manufacturer’s
instructions (IL-2, R&D Systems, Minneapolis, MN; TNF-, Bender
MedSystems, Vienna, Austria; IFN-, Endogen, Cambridge, MA). Cytokine
concentration was assayed in duplicate.
Transfections and luciferase assays
Transcriptional activity was measured using reporter gene assays
in transiently transfected Jurkat cells. The pBF-Luc (NF-B-LUC)
plasmid includes a trimer of the NF-B motif of the
H-2Kb gene upstream of the TK promoter (32). The
plasmid TNF--LUC contains a region 1311 bp upstream from the
transcriptional initiation site of human TNF- promoter (33). The
reporter constructs, NF-AT-LUC, containing three tandem copies of the
NF-AT binding site fused to the IL-2 minimal promoter, and the IL-2-LUC
plasmid, containing the region spanning from -326 to +45 of the human
IL-2 promoter, have been described previously (34). Both were a
generous gift of Dr. G. Crabtree (Stanford Medical School, Stanford,
CA). The plasmid TK-LUC contains the herpes simplex I thymidine kinase
promoter upstream the luciferase gene (35).
Jurkat cells were transfected with Lipofectin, as recommended by the manufacturer (Life Technologies). After transfection, cells were then treated with different stimuli as indicated for another 4–6 h, harvested by centrifugation, and lysed. Luciferase activity was measured in a luminometer following the instructions given in a luciferase kit assay (Promega, Madison, WI). Data are represented in relative luciferase units (RLU) or fold induction (observed experimental RLU/basal RLU in absence of any stimulus).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To analyze COX expression in T cells, RNA from Jurkat cells or
purified T lymphocytes was subjected to Northern blot and RT-PCR
analysis. As shown in Fig. 1
A,
very low levels of COX-1 and COX-2 mRNAs were detected by RT-PCR in
unstimulated Jurkat T cells. However, exposure of cells to the phorbol
ester PMA plus the Ca2+ ionophore A23187 (Ion), a
pharmacologic treatment known to mimic TCR/CD3 T cell activation (36),
led to a substantial induction of COX-2 mRNA. PMA by itself was able to
induce 2–3-fold COX-2 mRNA, whereas treatment with Ion alone did not
increase COX-2 expression, suggesting the involvement of protein kinase
C activation in COX-2 induction. A weak but significant induction of
COX-2 mRNA expression was also observed after stimulation with
immobilized anti-CD3. In contrast, COX-1 mRNA expression did not
change significantly after any of these treatments.
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B).
T lymphocytes were activated with pharmacological agents or with
immobilized anti-CD3 alone or combined with soluble CD28 Abs. This
latter treatment provides a costimulatory signal that induces a
substantial increase in T cell activation (37). COX-1 mRNA was also
observed in resting T lymphocytes, but its expression did not change
appreciably after stimulation. In contrast, COX-2 mRNA expression
increased after activation with PMA alone or in combination with Ion.
Treatment with immobilized anti-CD3 Abs alone also produced a
strong induction that was not further augmented by costimulation with
PMA or anti-CD28 Abs. The lower COX-2 mRNA response to immobilized
anti-CD3 in Jurkat cells compared with human T lymphocytes was
probably due to the low level of CD3 expression in our Jurkat cloned
line (data not shown). These results were confirmed by Northern blot
analysis. Thus, COX-1 and COX-2 steady-state levels of mRNA expression
both in Jurkat and T cells were below detection by Northern blotting, a
less sensitive technique. In agreement with the RT-PCR data, PMA + Ion
treatment increases several fold COX-2 mRNA expression both in Jurkat
and T cells. Human T lymphocytes stimulated with anti-CD3 plus
anti-CD28 increased as well COX-2 mRNA expression (Fig. 1, C and D). Together, these data indicate that T
cell activation induces COX-2 expression without affecting COX-1
levels.
Treatment with CsA, a potent immunosuppressor that inhibits T cell
activation and proliferation (38, 39), completely abolished COX-2 mRNA
induction in human T lymphocytes stimulated with anti-CD3 plus
anti-CD28 (Fig. 2A) or PMA
plus Ion (not shown) without affecting basal levels of COX-1
transcripts. Similar results were observed in Jurkat T cells, in which
CsA inhibited the weak PMA induction, whereas PMA + Ion induction was
reduced to PMA-induced levels (Fig. 2B). Results obtained by
Northern blot analysis were in agreement with those observed by RT-PCR
(Fig. 1, C and D).
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A).
CD69 mRNA expression, an immediate early gene in T cells, was induced
with a similar kinetic. Experiments performed in Jurkat cells
stimulated with PMA plus Ion showed a similar pattern of COX-2 mRNA
induction. Again, COX-1 expression remained unaffected, maintaining the
basal levels through the entire period studied (Fig. 3B).
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), indicating that the increase in
mRNA levels occurs mainly at the transcriptional level, requiring new
RNA synthesis. CHX stimulated the steady state levels of COX-2 mRNA in
Jurkat cells. In addition, CHX was able to superinduce the stimulation
of COX-2 transcription by PMA plus Ion. This pattern of expression,
independent of the requirement for new protein synthesis, identifies
COX-2 as a characteristic inducible early T cell activation gene
(40, 41).
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To address whether COX-2 mRNA induction was paralleled by COX-2
protein increase, Western blot analysis, using specific Abs against
COX-2 protein, was performed with extracts of T cells treated with
anti-CD3 plus anti-CD28 and Jurkat cells stimulated with PMA
plus Ion. COX 2 protein levels increased after T cell activation,
showing a pattern of induction similar to that of its mRNA, although
delayed. The COX-2 protein appeared later (6 h), reaching maximal
levels at 12–24 h (Fig. 5A).
A similar pattern of expression was observed with Jurkat T cells.
Treatment with CsA completely abrogated COX-2 protein induction in
these cells (Fig. 5B).
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).
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To address the physiologic role of COX-2 induction in T cells, we
studied the influence of NSAIDs, as COX inhibitors, on several T cell
activation events. Human T cells were activated by anti-CD3 plus
anti-CD28, and cell proliferation was measured in the presence of
Indomethacin, a classical COX inhibitor being 60 times more active
against COX-1 than COX-2 (42) or NS398, a newly described
COX-2-selective inhibitor being 1000-fold more efficient for COX-2
inhibition (20, 43). Treatment with NS398 diminished in a
concentration-dependent manner anti-CD3 plus anti-CD28-induced
T cell proliferation (an average of 70–80% at 100 µM in the several
experiments performed with different blood donors) (Table II). However, it failed to affect either
basal proliferation or cell viability, as measured by trypan blue dye
exclusion test (not shown). In contrast, Indomethacin at similar doses
did not affect T cell proliferation.
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-chain (an early gene, requiring
protein synthesis), and CD71 or transferrin receptor (a late gene) (1).
Thus, the interference with T cell activation can be monitored through
changes in the expression of markers of differentiation and
proliferation in the T cell surface. Anti-CD3 plus anti-CD28
treatment was able to induce cell surface expression of the IL-2R
-chain (CD25). This induction was significantly inhibited by NS398
at 100 µM (an average of 70–80% considering together both the
percentage of positive cells and the mean fluorescence intensity).
Transferrin receptor expression (CD71), a marker of cell proliferation,
was also inhibited by NS398 to a similar extent. Although Indomethacin
at the highest concentrations used showed a slight inhibition of CD25
and CD71, this effect was not statistically significant. Meanwhile,
COX-2-selective inhibition by NS398 did not significantly affect CD69
expression, further discarding nonspecific effects of this compound
(Table II).
As a consequence of cell activation, T cells produce a variety of
lymphokines such as IL-2, IL-5, IL-10, TNF-, and IFN-. The
coordinated production of these lymphokines is crucial for regulation
of the inflammatory response. We evaluated the immunomodulatory actions
of COX inhibitors on production of lymphokines by activated T cells
cultured in the presence or absence of Indomethacin or NS398. As shown
in Fig. 6, NS398, even at 10 µM,
strongly inhibited IFN- production by activated T lymphocytes. It
also substantially inhibited TNF-, and IL-2 production. However,
Indomethacin did not markedly alter either IL-2, TNF-, or IFN-
production upon activation.
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To evaluate whether the effect of COX-2-selective NSAIDs on IL-2
or TNF- expression was at the transcriptional level, we investigated
the regulation of their promoters in Jurkat cells transiently
transfected with TNF- or IL-2 promoter reporter plasmids TNF--LUC
and IL-2-LUC. After transfection, cells were activated with PMA plus
Ion in the presence or absence of COX inhibitors, and tested for
luciferase activity. COX-2-selective inhibitors NS398 and Celecoxib
efficiently inhibited PMA plus Ion-stimulated transcription from both
of those reporters in a dose-dependent manner (Fig. 7A). In contrast, Indomethacin
only slightly affected IL-2 promoter-dependent transcription at 100
µM. The effect of COX-2 inhibitors was not due to an interference
with the transcriptional or translational machinery or with the in
vitro activity of the luciferase enzyme, as the basal luciferase
activity in cells transfected with a plasmid containing the luciferase
gene cloned downstream of the TK promoter was not affected
substantially by any of these drugs (Fig. 7B).
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, IL-2, and IFN- depends on the
coordinate interactions among several transcription factors, including
members of the NF-AT and NF-B families. Thus, we evaluated the
effect of NSAIDs on the transcriptional activity of those factors, by
using reporter genes under the control of minimal promoters containing
binding sites for each of them. Activation by PMA plus Ion increased
the activity of these promoters. We found that NS398 and Celecoxib, but
not Indomethacin, effectively inhibited each of these promoters in a
dose-dependent manner (Fig. 8). Thus,
NS398 and Celecoxib treatment results in an inhibitory effect of
transcription factors that play general roles in the regulation of
inflammatory responses in most cell types. These observations suggest
that COX-2-selective NSAIDs might inhibit the expression of genes that
became up-regulated during T cell activation and differentiation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inducible expression of COX-2 in response to growth factors, tumor promoters, or cytokines has been previously and extensively described in several cell types, including fibroblasts, endothelial cells, and macrophages (15, 17), but not, to our knowledge, in T cells. The COX-1 isoform appears to be maintained at constant levels in virtually all of these cells, although stimulation with growth factors can result in a moderate increase (15, 17). On the other hand, COX-2 gene transcription after T cell activation fulfills all of the criteria for an immediate early T cell activation gene: it is rapidly inducible, independent on new protein synthesis, superinducible by CHX, and dependent on new RNA synthesis. The COX-2 gene has been previously defined as an inducible immediate early gene in other cell types (40, 46).
In addition, COX-2 induction was completely abolished by CsA, a potent
immunosuppressive drug that strongly inhibits the transcription of
early T cell activation genes, including a number of cytokine genes
such as IL-2, TNF-, IFN-, GM-CSF, as well as c-myc and
various related protooncogenes of the src family (38, 39, 47). As shown by Martin, et al. (48), CsA also effectively inhibits
COX-2 mRNA induction by IL-1 or TNF in rat mesangial cells.
To investigate the role of COX-2 on T cell activation events, we
treated T cells with NSAIDs. Thus, NS398, a highly selective COX-2
inhibitor (43), but not Indomethacin, a preferential COX-1 NSAID (42),
decreased T cell proliferation. Although it is generally accepted that
Indomethacin is a preferential but not selective inhibitor of COX-1,
the doses required to inhibit COX-2 vary with the assay conditions
(49). Thus, doses between 10 and 20 µM Indomethacin have been
reported to act efficiently as COX-1-specific inhibitor (50, 51). The
action of NS398 was accompanied by a decrease in IL-2R -chain (CD25)
and transferrin receptor (CD71) membrane surface expression. This
inhibition was presumably not an overall action unspecifically
affecting surface membrane expression or T cell activation, as the
induction of other cell surface Ags expressed in activated T cells such
as CD69 was not affected. NS398 inhibited efficiently IL-2, TNF-,
and IFN- production. Similar results were obtained with other
chemically unrelated COX-2-specific inhibitors such as Celecoxib (19)
(data not shown). The small inhibitory effect of Indomethacin observed
at the highest dose in some experiments may reflect their ability to
partially inhibit COX-2 enzymatic activity at that concentration. These
data support the hypothesis that highly COX-2-selective NSAIDs are
immunosuppressive agents. They inhibit not only T cell proliferation
and CD25 and CD71 expression, but also IL-2, TNF-, and IFN-
transcription.
We believe that both the expression of COX-2 and the effect of
COX-2-specific drugs in our system cannot be ascribed to the possible
contamination by monocytes/macrophages of our purified T cell
populations for several reasons: 1) the effects observed on normal T
cells can be mimicked in Jurkat T cells; 2) COX-2 induction (mrRNA and
protein) occurs rapidly in response to stimuli that specifically
activate T lymphocytes through the TCR/CD3 complex and were blocked by
CsA. Moreover, if the effects were at the level of macrophages, their
COX-2 would presumably produce PGE2 (10) that in
turn would decrease CD25 expression, IL-2, TNF-, and IFN-
production and cell proliferation (11, 12, 13, 14). Therefore, blocking of
COX-2-produced PGE2 by specific inhibitors will
produce opposite effects to those we observed, resulting in an
enhancement and not a decrease of T cell activation as we found. Thus,
other actions may account for COX-2 physiologic functions in the T cell
activation process.
In this regard, it is worth noting that some COX-2 inhibitors have been
shown to possess immunosuppressive effects and to inhibit cytokine
synthesis in other cell types in addition to their
anti-inflammatory properties. Thus, it is well known that
glucocorticoids, widely used as immunosuppressive and potent
anti-inflammatory drugs, also inhibit the induction of COX-2
expression in a variety of cell lines and in response to different
stimuli (52, 53). Tepoxalin, a cyclooxygenase and lipoxygenase
inhibitor, blocked neutrophil infiltration by diminishing up-regulation
of adhesion molecules E-selectin and Mac-1 and inhibited IL-2, IL-6,
IL-8, and IFN- induction in T cells (25, 54, 55). Moreover, a new
class of cytokine-suppressive anti-inflammatory drugs (CSAIDs) have
been defined as sharing both COX- and cytokine-inhibiting properties in
LPS-stimulated human monocytes (56, 57). Other NSAIDs, such as Tenidap,
Ibuprofen, and Naproxen, suppressed the proliferative response of T
cells to IL-2 (23). Furthermore, Tenidap inhibited IFN- production
and mRNA expression in T lymphocytes (24). Collectively, these data
suggest that the anti-inflammatory actions of NSAIDs will not be
only restricted to the inhibition of COX-dependent
PGE2 synthesis and support the hypothesis of the
immunomodulatory role of NSAIDs.
Activation of T cells through the TCR/CD3 complex triggers a program of gene expression that can be divided into three phases: immediate events (independent on new protein synthesis), early events (dependent on protein synthesis and preceding cell division), and late events (occurring after cell proliferation) (1). In response to TCR/CD3-transduced signals, T cells activate a number of immediate early activation genes, such as c-fos, c-myc, and members of the src family (1, 47). The production of these genes is presumably involved in controlling and initiating the cascade of transcriptional responses that lead to complete activation. Our results indicate that COX-2 belongs to this category of immediate early genes. Therefore, by analogy with other immediate early genes, COX-2 activation may participate in the cascade of events occurring after T cell activation. Because COX-2 and CD69 share the characteristic of immediate early genes, the lack of effect of COX-2 inhibitors on CD69 expression reinforces the fact that COX-2 actions specifically occur downstream in the early and late but not in the immediate early phase of T cell activation.
The mechanism of action of COX-2-inhibitory drugs appears to be related
to nuclear transcriptional activation. COX-2-specific inhibitors NS398
and Celecoxib effectively inhibited PMA plus Ion-stimulated
transcription from these promoters. IL-2, TNF-, and IFN-
activation take place through activation of transcription factors,
including members of the NF-AT and NF-B families (33, 58, 59). COX-2
inhibition also led to down-regulation of NF-AT- and NF-B-driven
transcription. These effects were specific because transcription,
translation, or activity of a TK-driven luciferase reporter was not
altered by any of these drugs. These observations suggest some general
effects of COX-2 inhibitors on the activity of TCR/CD3-driven signals
through pathways involving Ca2+/calcineurin or
NF-B. It has been shown previously that NF-AT and NF-B activation
are sensitive to CsA (60, 61). This suggests that calcineurin acts as a
common upstream signaling molecule. Moreover, our results indicate that
COX-2 induction is also sensitive to CsA, also suggesting the
requirement for calcineurin. However, COX-2 inhibition blocks NF-AT and
NF-B activation, placing COX-2 actions upstream, not downstream of
NF-AT. This apparent paradox reflects the fact that T cell activation
as well as NF-B and NF-AT activation are biphasic (60, 61). Both
factors are initially present in an inactive form in the cytoplasm of
resting T cells, and rapidly translocate to the nucleus upon
activation. Thus, it is impossible that COX-2 actions will be involved
in this initial phase because COX-2 protein is not present at this
moment. Rather, COX-2 actions will most likely occur at the second
phase of T cell activation. In support of this theory is the fact that
CD69, a NF-B-dependent immediate early gene, was not affected by
COX-2 inhibition. Both pathways share activation by numerous kinases
and phosphatases that might be targets of COX-2 inhibition in this
second phase. Together, these data emphasize the role of COX-2 in the T
cell signaling activation process.
On the other hand, our results may provide an explanation at the
molecular level for previous results with COX inhibitors. Thus, it has
been shown that aspirin and sodium salicylate inhibit NF-B-dependent
activation in Jurkat cells, thus decreasing dependent gene induction
(22). Interestingly, although both aspirin and sodium salicylate are
more effective COX-1 inhibitors, the doses used in these experiments
(1–10 mM) were high enough to also inhibit COX-2 activity (42).
Tepoxalin, a COX-2 inhibitor, also inhibits NF-B function and
activation (55). Taken together, several structurally unrelated COX-2
inhibitors, NS-398, Celecoxib, Tepoxalin, as well as Aspirin or
salicylate at high doses produce the same effects on T cells: blockade
of transcription factor NF-B-dependent induction. The most likely
explanation is that all of these effects derive from their inhibition
of COX-2 activity. COX-2 produces PGs within or in the nucleus
envelope, whereas COX-1 products are only located at the endoplasmic
reticulum (62). New undescribed COX-2 metabolites (or simply previously
described ones undetected because of their nuclear localization),
produced in the nucleus of activated T cells, may further regulate gene
transcription or other nuclear events during cell activation.
However, other mechanisms cannot be discarded. Evidence of a mechanism
involving gene regulation by eicosanoids through nuclear receptors of
the PPAR family has been described (26, 27, 63). Thus, PGs of the J
series regulate gene expression of proinflammatory genes at the
promoter level in macrophages by binding to PPAR- transcription
factor (64). In agreement with a role of COX-2 metabolites in gene
transcription is the fact that COX-2 overexpression controls
mitogenesis, apoptosis, carcinogenesis, and even angiogenesis at the
transcription level in other cell types (65, 66, 67), further supporting an
important role of COX-2 in many important aspects of cell activation.
Alternatively, COX might play an important role through its peroxidase
activity on T lymphocyte function and activation, because transcription
factors with a key role in this process such as NF-B can be
activated by COX-1, apparently using intracellular reactive oxygen
intermediates as second messengers (68). It has been shown recently
that aspirin and salicylate inhibit NF-B activation through binding
to the IB kinase ß (IK-ß) (69). Thus, it is possible that new
targets for NS398 and Celecoxib may account for some of their actions
in addition to COX-2 enzymatic inhibition. Together, these data suggest
that COX-2 actions may not only be exerted through synthesis of
classical PGs, and new mechanisms of action that play a key role in
controlling activation processes remain to be elucidated.
In summary, this study provides the first evidence that transcriptional up-regulation of COX-2 isoenzyme occurs after T cell activation, and suggests functional implications of COX-2 activity in this process. More importantly, our results suggest that NSAIDs with COX-2 selectivity may be attractive anti-inflammatory drugs, as they possess additional anti-inflammatory properties to their selective down-regulation of PG production, such as regulation of the immune activation and the production of proinflammatory cytokines by T cells. This may have profound implications for NSAIDs therapy and the development of new COX-2-specific inhibitors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Manuel Fresno, Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: AA, arachidonic acid; ActD, actinomycin D; CHX, cycloheximide; COX, cyclooxygenase; CsA, cyclosporin A; Ion, calcium ionophore A23187; LUC, luciferase; NSAID, nonsteroidal anti-inflammatory drugs; PPAR, peroxisome proliferator-activated factor; RLU, relative luciferase unit.
Received for publication September 25, 1998. Accepted for publication April 9, 1999.
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References |
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) or COX-1 (•)
mRNA to GAPDH mRNA.
70 kDa) is detected by this Ab in the control lane (labeled COX-2)
that contains purified ovine COX-2.
