Effect of acetylsalicylic acid on endogenous Ikappa B kinase activity in lung epithelial cells

Chul-Gyu Yoo1,2,3, Seunghee Lee1,2, Choon-Taek Lee1,2,3, Young Whan Kim1,2,3, Sung Koo Han1,2,3, and Young-Soo Shim1,2,3

1 Department of Internal Medicine, Seoul National University College of Medicine, and 3 Lung Institute, Seoul National University Medical Research Center, Chongno-Gu, Seoul 110-799; and 2 Clinical Research Institute, Seoul National University Hospital, Chongno-Gu, Seoul 110-744, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The anti-inflammatory effect of acetylsalicylic acid (ASA) has been thought to be secondary to the inhibition of prostaglandin synthesis. Because doses of ASA necessary to treat chronic inflammatory diseases are much higher than those needed to inhibit prostaglandin synthesis, a prostaglandin-independent pathway has been emerging as the new anti-inflammatory mechanism of ASA. Here, we examined the effect of ASA on the interleukin (IL)-1beta - and tumor necrosis factor (TNF)-alpha -induced proinflammatory cytokine expression and evaluated whether this effect is closely linked to the nuclear factor (NF)-kappa B/Ikappa B-alpha pathway. A high dose of ASA blocked IL-1beta - and TNF-alpha -induced TNF-alpha and IL-8 expression, respectively. ASA inhibited TNF-alpha -induced activation of NF-kappa B by preventing phosphorylation and subsequent degradation of Ikappa B-alpha in a prostanoid-independent manner. TNF-alpha -induced activation of Ikappa B kinase was also suppressed by ASA pretreatment. These observations suggest that the anti-inflammatory effect of ASA in lung epithelial cells may be due to suppression of Ikappa B kinase activity, which thereby inhibits subsequent phosphorylation and degradation of Ikappa B-alpha , activation of NF-kappa B, and proinflammatory cytokine expression in lung epithelial cells.

nuclear factor-kappa B; interleukin-8


    INTRODUCTION
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INTRODUCTION
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OVER THE LAST DECADE, many studies of basic biological characteristics of inflammation and tissue injury have implicated proinflammatory cytokine-mediated tissue injury in the pathogenesis of a wide variety of inflammatory disorders including sepsis, acute respiratory distress syndrome, and multiorgan dysfunction syndrome. As a result, anti-inflammatory agents, which inhibit the expression of proinflammatory cytokines, have been tried as the specific therapy for these diseases (4).

Acetylsalicylic acid (ASA) is a nonsteroidal anti-inflammatory drug (NSAID) used in the treatment of many inflammatory diseases. It is rapidly deacetylated to salicylate in the intact organism. Its ability to inhibit arachidonic acid metabolites by blocking cyclooxygenase (COX) and prostaglandin H synthase has been regarded as the main anti-inflammatory mechanism. However, doses of ASA necessary to treat chronic inflammatory diseases are much higher than those needed to inhibit prostaglandin synthesis (21, 25). In addition, nonacetylated salicylates, which do not interfere with prostaglandin synthesis, are still effective anti-inflammatory agents when used in high doses (21, 25). These findings have led to the speculation that the anti-inflammatory effect of ASA may be mediated by a prostaglandin-independent pathway.

Nuclear factor (NF)-kappa B is a ubiquitous inducible transcription factor involved in immune, inflammatory, stress, and developmental processes. It is sequestered in the cytoplasm in an inactive state by association with the inhibitory molecule Ikappa B-alpha . NF-kappa B is rapidly activated in response to various stimuli including viral infection, lipopolysaccharide, ultraviolet (UV) irradiation, and proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta (2, 3, 11). TNF-alpha leads to the sequential activation of the downstream NF-kappa B-inducing kinase (NIK) and the recently isolated TNF-alpha -inducible Ikappa B kinase (IKK) complex (9, 15, 23, 26, 29). When activated, IKK directly phosphorylates Ser32 and Ser36 of Ikappa B-alpha , triggering ubiquitination at Lys21 and Lys22 and rapid degradation of Ikappa B-alpha in 26S proteasomes (2, 3, 11). This process liberates NF-kappa B, allowing it to translocate to the nucleus. In the nucleus, NF-kappa B binds to its cognate kappa B site and transactivates the downstream genes. Most genes for inflammatory mediators such as TNF-alpha , IL-2, IL-6, IL-8, lymphotoxin, granulocyte-macrophage colony-stimulating factor, interferon-beta , and adhesion molecules have kappa B sites in the 5'-flanking region (2, 3, 11).

Recent reports (14, 16, 20, 24) suggest that high doses of salicylates show an anti-inflammatory effect through the inhibition of NF-kappa B activation in monocytic, lymphocytic, and endothelial cells. However, the anti-inflammatory effect of ASA and its mechanism of action in lung epithelial cells are poorly understood. In the present study, we investigated the effect of ASA on proinflammatory cytokine expression and evaluated whether this effect is closely linked to NF-kappa B/Ikappa B-alpha regulation in lung epithelial cells. First, we found that a high dose of ASA blocked TNF-alpha -induced IL-8 mRNA and protein expression. Second, ASA pretreatment inhibited TNF-alpha -induced activation of NF-kappa B by preventing the degradation of Ikappa B-alpha . Finally, TNF-alpha -induced activation of endogenous IKK and subsequent phosphorylation of Ikappa B-alpha were suppressed by ASA pretreatment. These observations suggest that the anti-inflammatory effect of ASA in lung epithelial cells may be due to the blocking of Ikappa B-alpha phosphorylation by suppressing IKK activity, thereby inhibiting subsequent degradation of Ikappa B-alpha , activation of NF-kappa B, and proinflammatory cytokine expression.


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Cell culture. BEAS-2B cells, representing normal human bronchial epithelial cells, were maintained as a monolayer in keratinocyte growth medium (Clonetics, Walkersville, MD), and A549 cells, representing type II alveolar epithelial cells, were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 60 µg/ml of penicillin, and 100 µg/ml of streptomycin at 37°C under 5% CO2.

Reagents. Recombinant human TNF-alpha and an ELISA kit for IL-8 were purchased from R&D Systems (Minneapolis, MN). A stock solution of TNF-alpha was prepared in distilled water, and aliquots were stored at -70°C until used. Rabbit polyclonal anti-Ikappa B-alpha , anti-p65, and anti-IKK-alpha antibodies and recombinant glutathione S-transferase (GST)-Ikappa B-alpha were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-phosphorylated Ikappa B-alpha antibody (Ser32) was supplied by New England Biolabs (Beverly, MA). Goat anti-rabbit secondary antibody conjugated with horseradish peroxidase and T4 polynucleotide kinase were purchased from Promega (Madison, WI). Rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody was obtained from Jackson ImmunoResearch (West Grove, PA). Protein G Sepharose beads and an enhanced chemiluminescence kit were supplied by Amersham Pharmacia Biotech (Uppsala, Sweden). Protease inhibitors were obtained from Roche (Mannheim, Germany). ASA, indomethacin, and prostaglandin E2 were obtained from Sigma (St. Louis, MO). The proteasome inhibitor N-carbobenzoxyl-Leu-Leu-Leu-leucinal (MG-132) was purchased from the Peptide Institute (Osaka, Japan). TRIzol reagent was obtained from GIBCO BRL (Life Technologies, Gaithersburg, MD). [alpha -32P]dCTP and [gamma -32P]ATP were supplied by ICN Pharmaceuticals (Costa Mesa, CA). A random-priming kit was purchased from Stratagene (La Jolla, CA).

Northern blot analysis. Total cellular RNA was isolated with TRIzol reagent. Equal amounts of total RNA (20 µg/lane) from each sample were loaded into each lane of 1.0% agarose-2% formaldehyde gels and capillary transferred to nylon membrane. The RNA was cross-linked to the nylon membrane by 1,500-J UV irradiation in a UV cross-linker (Stratagene). The human cDNA for IL-8 was radiolabeled with [alpha -32P]dCTP with a random-priming kit. After prehybridization of the membranes for 2 h at 45°C in hybridization buffer, radiolabeled cDNA probe (1 × 106 counts · min-1 · ml-1 final concentration) was added and incubated overnight at 45°C. The membranes were then washed at 45, 50, and then 55°C. The membranes were exposed to X-ray film in a cassette with an intensifying screen at -70°C.

IL-8 ELISA. Cells (1 × 104) were grown in 96-well culture plates in equal numbers. The supernatants were collected and stored at -70°C until analyzed. IL-8 concentrations were quantified with an ELISA kit according to the manufacturer's specifications.

Western blot analysis. Cytoplasmic, nuclear, and whole cell extracts were prepared as previously described (28). Twenty micrograms of protein were resolved by 10% SDS-PAGE and transferred to nitrocellulose. The membranes were blocked with 5% skim milk-PBS-0.1% Tween 20 for 1 h before overnight incubation at room temperature with rabbit polyclonal anti-p65 antibody, anti-Ikappa B-alpha antibody, or antibody specific for phosphorylated Ikappa B-alpha diluted 1:1,000 in 5% skim milk-PBS-0.1% Tween 20. The membranes were washed three times in 1× PBS-0.1% Tween 20 and incubated with goat-anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:2,000 in 5% skim milk-PBS-0.1% Tween 20 for 1 h. After successive washes, the membranes were developed with an enhanced chemiluminescence kit.

Immunofluorescent staining for NF-kappa B. The cells grown in two-well chamber slides were fixed and permeabilized as previously described (28). The cells were incubated with rabbit polyclonal anti-p65 antibody diluted 1:100 in 1% BSA for 30 min. The cells were incubated with rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody diluted 1:100 in 1% BSA for 30 min. After being mounted with 50% glycerol, the slides were analyzed with a fluorescence light microscope.

Electrophoretic mobility shift assays. NF-kappa B DNA binding activity was assessed as previously described (28). Briefly, nuclear extracts were incubated for 20 min at room temperature with a radiolabeled NF-kappa B consensus sequence in the kappa  light chain enhancer in B cells (5'-AGTTGAGGGGACTTTCCCAGGC-3'). In competition experiments, a 50-fold molar excess of unlabeled oligonucleotide was added to the binding reaction. In supershift experiments, 0.4 µg of anti-p65 or anti-p50 antibody was added and allowed to react for 45 min at room temperature. DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide gels. The gels were dried and autoradiographed at -70°C.

IKK assay. IKK activity was assessed with an in vitro kinase assay as previously described (28). In brief, the IKK complex was immunoprecipitated with an anti-IKK-alpha antibody diluted 1:100. The immunoprecipitates were incubated at 30°C for 30 min in a kinase buffer containing 0.5 µg of GST-Ikappa B-alpha (containing amino acids 1-317) and 10 µCi of [gamma -32P]ATP. Kinase reaction products were subjected to SDS-PAGE in 10% gels followed by transfer to a nitrocellulose membrane and autoradiography.


    RESULTS
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INTRODUCTION
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DISCUSSION
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ASA blocks IL-1beta - and TNF-alpha -induced proinflammatory cytokine expression. To determine whether ASA shows anti-inflammatory effects in lung epithelial cells, we first analyzed the effect of ASA on proinflammatory cytokine expression. To evaluate whether ASA inhibits cytokine production dose dependently, A549 cells were pretreated with medium or various amounts of ASA (2.5, 5, 10, or 20 mM) for 2 h and then stimulated with IL-1beta or TNF-alpha . IL-8 concentrations in the culture supernatants were assayed by ELISA after 18 h of TNF-alpha stimulation. TNF-alpha increased IL-8 production in the absence of ASA. Both IL-1beta - and TNF-alpha -induced IL-8 production were reduced by pretreatment with a high dose of ASA (Fig. 1A). To evaluate whether the reduction in TNF-alpha -induced IL-8 production was due to the decrease in mRNA expression, the cells were pretreated with 20 mM ASA for 2 h and then stimulated with IL-1beta or TNF-alpha for 4 h. IL-1beta -induced TNF-alpha and IL-1beta - and TNF-alpha -induced IL-8 mRNA expression were assayed by Northern blot analysis. Although TNF-alpha mRNA was hardly detectable in untreated cells, IL-1beta induced a marked increase in TNF-alpha mRNA 4 h after stimulation, and this increase was blocked completely in the presence of ASA (Fig. 1B). Both IL-1beta - and TNF-alpha -induced IL-8 mRNA expression were also suppressed by ASA pretreatment (Fig. 1B). To exclude the possibility that this effect of ASA is due to its cytotoxicity, the cells were incubated in the presence of 2.5, 5, 10, or 20 mM ASA for 2.5 h. Cell viability was evaluated by MTT assay. Cell viability did not change in both cells at all doses used (data not shown). These observations indicate that ASA shows anti-inflammatory effects in lung epithelial cells by inhibiting proinflammatory cytokine production.


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Fig. 1.   Acetylsalicylic acid (ASA) blocked interleukin (IL)-1beta - and tumor necrosis factor (TNF)-alpha -induced proinflammatory cytokine expression. A: dose-dependent effect of ASA on the production of IL-8 by IL-1beta (left) or TNF-alpha (right). A549 cells were treated with medium alone or indicated doses of ASA for 2 h and then stimulated with IL-1beta (5 ng/ml) or TNF-alpha (5 ng/ml) for 18 h in the continued presence (+) and absence (-) of ASA. The concentrations of IL-8 in supernatant fluid were quantitated by ELISA. Data are means ± SD from 3 different experiments. B: effect of ASA on proinflammatory cytokine mRNA expression. A549 cells were treated with medium alone or ASA for 2 h and then stimulated with IL-1beta or TNF-alpha for 4 h. IL-1beta -induced TNF-alpha and IL-1beta - and TNF-alpha -induced IL-8 mRNA expression were assayed by Northern blot analysis. Results are representative of 3 different experiments.

NF-kappa B activation is inhibited by ASA. Because most of the proinflammatory cytokine genes including IL-8 contain kappa B-binding motifs in their promoter regions, we questioned whether the inhibition of proinflammatory cytokine expression by ASA is due to the blocking of TNF-alpha -induced activation of NF-kappa B. NF-kappa B activation was assayed by two approaches: one measured the nuclear translocation of NF-kappa B and the other assessed the NF-kappa B-DNA binding activity by electrophoretic mobility shift assay. The expression of NF-kappa B was assayed by Western blot analysis for the p65 subunit of NF-kappa B in cytoplasmic and nuclear extracts from cells stimulated with TNF-alpha in the presence and absence of ASA. Although the majority of p65 was located in the cytoplasmic fraction in the basal state, p65 increased in the nuclear fraction 30 min after TNF-alpha stimulation, which was completely blocked by ASA pretreatment (Fig. 2A). Total cellular expression of p65 was not affected by ASA pretreatment (Fig. 2A). We next investigated the subcellular localization of NF-kappa B by immunofluorescent staining. There was a strong nuclear staining of p65 30 min after stimulation with TNF-alpha in both BEAS-2B and A549 cells compared with the cytoplasmic distribution in unstimulated cells. This nuclear translocation of p65 by TNF-alpha was blocked by ASA pretreatment as demonstrated by the cytoplasmic staining pattern (Fig. 2B). We next evaluated the effect of ASA on the NF-kappa B-DNA binding activity by electrophoretic mobility shift assay. Nuclear extracts from TNF-alpha -stimulated cells had more active NF-kappa B available to bind to the kappa B probe compared with extracts from untreated cells. This TNF-alpha -induced increase in NF-kappa B-DNA binding activity was inhibited by 20 mM ASA (Fig. 2C). When a 50-fold molar excess of unlabeled double-strand NF-kappa B oligonucleotide was added to the binding reaction, the retarded band disappeared, suggesting the specificity of binding. Supershift assay showed the presence of the p50 and p65 subunits of NF-kappa B (Fig. 2C). These results indicate that suppression of proinflammatory cytokine expression by ASA is due to the blockade of NF-kappa B activation.


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Fig. 2.   Nuclear factor (NF)-kappa B activation was inhibited by ASA pretreatment. A: effect of ASA on TNF-alpha -induced nuclear translocation of NF-kappa B. A549 cells were treated with ASA for 2 h and then stimulated with TNF-alpha for 30 min. Cytoplasmic (C), nuclear (N), and total cellular (T) protein extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. NF-kappa B subunit p65 was detected by Western blot analysis. B: effect of ASA on subcellular localization of NF-kappa B. Cells in chamber slides were treated with medium alone or ASA for 2 h and then stimulated with TNF-alpha for 30 min. Cells were fixed and permeabilized for 5 min. Immunofluorescent staining for p65 was performed with p65 antibody followed by rhodamine-conjugated detection antibody. C: effect of ASA on NF-kappa B-DNA binding activity. Cells were stimulated with TNF-alpha for 30 min with indicated doses of ASA. Nuclear extracts were made and subjected to electrophoretic mobility shift assays with kappa B site DNA probe as described in METHODS. Results are representative of 3 different experiments.

ASA suppresses Ikappa B-alpha degradation in a prostanoid-independent manner. Because NF-kappa B exists as an inactive form bound to the inhibitory protein Ikappa B-alpha in the cytoplasm, the degradation of Ikappa B-alpha must occur in order for NF-kappa B to translocate to the nucleus. We next analyzed the effect of ASA on the IL-1beta - and TNF-alpha -induced degradation of Ikappa B-alpha . IL-1beta - and TNF-alpha -induced degradation of Ikappa B-alpha was blocked by high doses of ASA (Fig. 3A). ASA is known to be a weak inhibitor of COX activity in respiratory epithelial cells. To evaluate whether the inhibition of COX activity stabilizes Ikappa B-alpha , we examined the effect of indomethacin, which is a potent COX inhibitor, on the TNF-alpha -induced degradation of Ikappa B-alpha . Ikappa B-alpha degradation by TNF-alpha was not blocked by indomethacin pretreatment at all doses used (10, 50, 100, and 500 µM; Fig. 3B). To evaluate whether ASA stabilizes Ikappa B-alpha in a prostanoid-dependent manner, we next examined the effect of exogenously applied prostaglandin E2 on the Ikappa B-alpha stabilizing effect of ASA. Exogenously applied prostaglandin E2 did not reduce the blocking effect of ASA on the TNF-alpha -induced degradation of Ikappa B-alpha (Fig. 3C). These observations suggest that ASA-induced blocking of NF-kappa B activation is due to the stabilization of Ikappa B-alpha in a prostanoid-independent manner.


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Fig. 3.   ASA stabilized Ikappa B-alpha in a prostanoid-independent manner. A: only high doses of ASA suppressed IL-1beta - and TNF-alpha -induced Ikappa B-alpha degradation dose dependently. A549 cells were treated with medium alone or indicated doses of ASA for 2 h and then stimulated with IL-1beta (A, top) or TNF-alpha (A, bottom) for 30 min. Ikappa B-alpha was detected by Western blot analysis. B: indomethacin (IND) did not block TNF-alpha -induced degradation of Ikappa B-alpha . BEAS-2B cells were pretreated with indicated doses of indomethacin for 2 h and then stimulated with TNF-alpha for 30 min. Ikappa B-alpha was detected by Western blot analysis. C: effect of exogenous prostaglandin E2 (PGE2) on the Ikappa B-alpha stabilizing effect of ASA. BEAS-2B cells were pretreated with ASA and indicated doses of PGE2 for 2 h and then stimulated with TNF-alpha for 30 min. Ikappa B-alpha was detected by Western blot analysis. Results are representative of 3 different experiments.

Ikappa B-alpha phosphorylation is blocked by ASA. Ikappa B-alpha degradation by a proteasome-dependent pathway is preceded by phosphorylation on two serine residues (Ser32 and Ser36) and ubiquitination. To address the mechanism involved in the stabilization of Ikappa B-alpha by ASA, we examined IL-1beta - and TNF-alpha -induced phosphorylation of Ikappa B-alpha by Western blot analysis. The cells were pretreated with the proteasome inhibitor MG-132, which allows the phosphorylated Ikappa B-alpha to accumulate in the cell. As previously reported (8, 17, 22), MG-132 pretreatment stabilized the phosphorylated Ikappa B-alpha in response to IL-1beta and TNF-alpha , which was detectable as a slower migrating band (Fig. 4A). These slower migrating bands disappeared after ASA pretreatment (Fig. 4A). Because these slower migrating bands disappeared when extracts were incubated with calf intestinal phosphatase, they were considered to be phosphorylated Ikappa B-alpha (data not shown). This was further confirmed by immunoblotting with an antibody specific to phosphorylated Ikappa B-alpha on Ser32 (Fig. 4B). These observations suggest that stabilization of Ikappa B-alpha by ASA may be due to the inhibition of Ikappa B-alpha phosphorylation.


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Fig. 4.   Ikappa B-alpha phosphorylation was blocked by ASA. A: BEAS-2B cells were treated with the proteasome inhibitor MG-132 (20 µM) with and without ASA for 2 h and then stimulated with IL-1beta or TNF-alpha for 30 min. Western blot analysis for Ikappa B-alpha was carried out with whole cell extracts with a rabbit polyclonal anti-Ikappa B-alpha antibody. B: whole cell extracts from cells treated in A were assayed for phosphorylated Ikappa B-alpha (p-Ikappa B-alpha ) by Western blot analysis with an antibody specific to phosphorylated Ikappa B-alpha on Ser32. Results are representative of 3 different experiments.

ASA blocks activation of IKK. Cytokine-induced Ikappa B-alpha phosphorylation is mediated by the IKK complex. To evaluate whether the decrease in phosphorylated Ikappa B-alpha in ASA-treated cells is due to the inhibition of IKK activity, the effect of ASA on IKK activity was assayed with GST-Ikappa B-alpha as a substrate after TNF-alpha stimulation with various doses of ASA. TNF-alpha induced a marked increase in phosphorylated GST-Ikappa B-alpha after 5 and 10 min of stimulation in BEAS-2B and A549 cells, respectively, which implies activation of the IKK complex. TNF-alpha -induced phosphorylation of Ikappa B-alpha was completely blocked by ASA pretreatment at doses of 10 mM in BEAS-2B cells and 20 mM in A549 cells, suggesting different sensitivities to ASA in different cell lines (Fig. 5). This inhibition of IKK activity by ASA was not due to a decrease in IKK-alpha protein levels because immunoblot analysis demonstrated comparable IKK-alpha expression at all conditions (data not shown). These results indicate that inhibition of Ikappa B-alpha phosphorylation by ASA may be through the inhibition of IKK activation.


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Fig. 5.   ASA blocked activation of Ikappa B kinase (IKK). BEAS-2B and A549 cells were pretreated with indicated doses of ASA for 2 h and then stimulated with TNF-alpha for 5 and 10 min, respectively. IKK complex was immunoprecipitated with an anti-IKK-alpha antibody, and IKK assays were performed as described in METHODS. GST, glutathione S-transferase. Results are representative of 3 different experiments.


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Salicylates have been shown to inhibit the transcription of genes such as adhesion molecules and inducible nitric oxide synthase, which are involved in the inflammation process (10, 20). Because proinflammatory cytokine-mediated tissue injury has been implicated in the pathogenesis of a wide variety of inflammatory disorders and transcription of most proinflammatory cytokine genes is dependent on NF-kappa B activation, it is very likely that the anti-inflammatory effect of salicylates is closely related to the suppression of proinflammatory cytokine expression. Although ASA and sodium salicylates inhibit NF-kappa B activation in monocytic, lymphocytic, and endothelial cells (14, 16, 20, 24), TNF-alpha -induced activation of NF-kappa B was not blocked by salicylates in cultured cardiac fibroblasts (10). The effect of salicylates on the activation of NF-kappa B differs according to cell type. At present, few data about the anti-inflammatory effect of ASA and its mechanism of action in lung epithelial cells have been presented, whereas the importance of the role of lung epithelial cells in lung inflammation is increasing.

In this study, we demonstrated that ASA inhibited IL-1beta - and TNF-alpha -induced proinflammatory cytokine expression by blocking NF-kappa B activation in lung epithelial cells. This effect of ASA can be generalized to respiratory epithelial cells because the same effects were observed in both BEAS-2B and A549 cells, which represent bronchial and alveolar epithelial cells, respectively. Considering the important role of proinflammatory cytokines in the inflammatory process, this result suggests an anti-inflammatory effect of ASA in lung epithelial cells. We also found that blocking of NF-kappa B activation was due to the stabilization of Ikappa B-alpha . This result coincides with previous reports in monocytic, lymphocytic, and endothelial cells (14, 16, 20, 24).

Prostanoids are important mediators of airway inflammation, and their synthesis is mediated by COX. NSAIDs, which potently inhibit COX activity, reduced IL-8 production in a prostanoid-dependent manner in airway smooth muscle cells (18), and a study (19) has shown that inhaled NSAIDs are protective in airway inflammation. Because ASA is known to inhibit COX activity, we examined whether the Ikappa B-alpha stabilizing effect of ASA is prostanoid dependent. Indomethacin, which potently inhibits COX-induced prostaglandin E2 production, did not block TNF-alpha -induced degradation of Ikappa B-alpha , and the Ikappa B-alpha stabilizing effect of ASA was not overcome by exogenously applied prostaglandin E2. The most likely explanation for this result is that the anti-inflammatory effect of ASA is prostanoid independent in lung epithelial cells.

Because NF-kappa B is sequestered in the cytoplasm by Ikappa B-alpha , activation of NF-kappa B requires degradation of Ikappa B-alpha . The first step of degradation involves phosphorylation of Ikappa B-alpha by the IKK complex. The IKK complex is made up of several kinases including IKK-alpha and IKK-beta , and it requires phosphorylation by NIK to become activated. Phosphorylated Ikappa B-alpha undergoes ubiquitination and finally degradation through a proteasome pathway. Because TNF-alpha -induced phosphorylation of Ikappa B-alpha was not observed in the presence of the proteasome inhibitor in this study, the Ikappa B-alpha stabilizing effect of ASA is likely to occur at the level of Ikappa B-alpha phosphorylation by either inhibition of IKK activity or activation of phosphatase. Because our immune complex kinase assay showed that ASA suppressed endogenous IKK activity, it is likely that ASA blocks Ikappa B-alpha phosphorylation by suppressing IKK activation rather than by activating a phosphatase.

The TNF-alpha - and IL-1beta -induced NF-kappa B/Ikappa B signaling pathway involves distinct pathways. TNF-alpha stimulation recruits TNF receptor-associated factor (TRAF)-2 and the receptor-interacting protein (12, 13), whereas IL-1beta uses the IL-1 receptor (IL-1R) accessory protein and the IL-1R-associated kinase to transmit signals to TRAF-6 (6, 7). The TNF-alpha and IL-1beta pathways converge on NIK to activate the IKK complex. Thus the target to block cytokine-induced degradation of Ikappa B-alpha could be the receptor-interacting protein, TRAF-2, TRAF-6, NIK, or IKK. However, because ASA pretreatment in this study blocked both TNF-alpha - and IL-1beta -induced phosphorylation of Ikappa B-alpha by inhibiting the activation of IKK, it seems likely that ASA interferes with a common signal upstream or parallel to IKK.

It has been reported that in vitro treatment with salicylates or ASA inhibited IKK-beta but failed to affect IKK-alpha (27). In this study, our results demonstrated that treatment of intact cells with ASA inhibited TNF-alpha -induced endogenous IKK activity. Although we used anti-IKK-alpha antibody for immunoprecipitation, it cannot be concluded that ASA blocks the function of endogenous IKK-alpha because IKK-alpha and IKK-beta form a complex and IKK-beta can be coimmunoprecipitated with anti-IKK-alpha antibody.

The concentrations of ASA that blocked TNF-alpha -induced IKK activation in our experiments were 10 and 20 mM in BEAS-2B and A549 cells, respectively, which is much higher than the usual therapeutic serum concentration. Only high concentrations of ASA blocked nuclear translocation of NF-kappa B in endothelial cells (20, 24). Therefore, it seems likely that this in vitro effect of ASA cannot be applied to the anti-inflammatory effect in vivo. Salicylates accumulate in the mildly acidic environments prevailing at sites of inflammation (1, 5, 25). Salicylates are uncharged at low pH and can readily cross membranes (5). Therefore, the local concentrations of ASA could be much higher than those of serum and would be sufficient to suppress IKK activation and subsequent NF-kappa B-dependent expression of proinflammatory cytokines.

In this study, we have shown that high doses of ASA inhibit proinflammatory cytokine production by blocking NF-kappa B activation in lung epithelial cells. We demonstrated that this inhibitory effect of ASA on NF-kappa B activation is secondary to the stabilization of Ikappa B-alpha by blocking the phosphorylation of Ikappa B-alpha and its subsequent degradation. This blocking of Ikappa B-alpha phosphorylation by ASA was due to the inhibition of IKK activity. Because proinflammatory cytokines function in redundant and overlapping ways through the so-called cytokine "cascade" or "network," it would be necessary to modulate the entire cytokine network at the same time to achieve an anti-inflammatory response. Because transcription of most proinflammatory cytokine genes is regulated by NF-kappa B activation, our data showing that inhibition of IKK activation by ASA resulted in the stabilization of Ikappa B-alpha and inhibition of NF-kappa B activation suggest that the IKK complex could be an excellent molecular target for a new anti-inflammatory therapy.


    ACKNOWLEDGEMENTS

We thank Dr. Sarang Kim for proofreading this manuscript.


    FOOTNOTES

This work was supported by a 1998 National Research and Development Program (Ministry of Science and Technology).

Address for reprint requests and other correspondence: C. G. Yoo, Dept. of Internal Medicine, Seoul National Univ. College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea (E-mail: cgyoo{at}snu.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 August 2000; accepted in final form 11 September 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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