Abstract
Although the precise mechanisms by which steroids mediate their therapeutic effects remain unknown, steroids have been reported to abrogate cytokine-induced activation of the transcription factor NF-κB. In some cell types, NF-κB activation is necessary to regulate cytokine-mediated cellular functions. However, compelling evidence suggests that the steroid inhibition of NF-κB is complex and cell specific. Using EMSA, we show that stimulation with TNF-α or IL-1β induces NF-κB DNA-binding activity in human airway smooth muscle cells. TNF-α and IL-1β also increased luciferase activity in airway smooth muscle cells transfected with a reporter plasmid containing κB enhancer elements. Cytokines activated NF-κB by rapidly degrading its cytosolic inhibitor IκBα, which was then regenerated after 60 min. Cytokine-mediated IκBα reappearance was completely blocked by the protein synthesis inhibitor cycloheximide. Inhibition of cytokine-mediated IκBα proteolysis using the protease inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and N-acetyl-l-leucinyl-l-leucinyl-norleucinal also inhibited cytokine-mediated early expression of ICAM-1. Although dexamethasone partially inhibited IL-1β- and TNF-α-induced up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on cytokine-induced ICAM-1 expression at 18–24 h. In addition, neither cytokine-induced degradation or resynthesis of IκBα nor NF-κB DNA-binding activity were affected by dexamethasone. In cells transfected with the luciferase reporter, dexamethasone did not affect TNF-α-induced NF-κB-dependent transcription. Interestingly, cytokine-mediated expression of cyclooxygenase-2 was completely abrogated by dexamethasone at 6 h. Together, these data demonstrate that cytokine-mediated NF-κB activation and ICAM-1 expression involve activation of a steroid-insensitive pathway.
The Rel/NF-κB transcription factors regulate myriad cellular processes that include cell proliferation, apoptosis, and differentiation (1, 2). Rel/NF-κB regulates transcription by direct binding to decameric sequences (κB motifs) located in the promoters and enhancers of a variety of viral and cellular genes, particularly those involved in inflammatory responses. In many cell types, Rel/NF-κB exists in the cytoplasm as an inactive form through association with inhibitory proteins called inhibitors of NF-κB (IκBs)3 (reviewed in Refs. 3 and 4). In the unstimulated cell, IκB masks the NF-κB nuclear localization signal. Treatment of cells with mitogens, growth factors, or cytokines promotes rapid nuclear translocation of Rel/NF-κB by a mechanism in which IκB kinases, IKK-1 and IKK-2, phosphorylate conserved serine residues within the amino terminus of the IκBα and IκBβ, which then targets IκB for ubiquitin-dependent degradation by the 26S proteasome (1, 2, 5). NF-κB inactivation occurs either by replenishing pools of IκBα by NF-κB-dependent stimulation of de novo synthesis of IκBα or by the regulated phosphorylation of IκBβ, whose expression is NF-κB-independent. Despite research that has markedly improved our understanding of the mechanisms by which cytokines activate NF-κB, arguably the most important unanswered questions still focus on identifying the upstream signaling events coupling cytokine receptors to activation of NF-κB.
Compelling evidence suggests that the role of NF-κB in cytokine signaling and the attendant cellular responses are highly cell- and tissue-specific (1, 2, 6). In hemapoietic cells, TNF-α activates NF-κB and AP-1 and induces apoptosis in an NF-κB-dependent manner (1). In many different cell types, including lymphocytes, NF-κB also inhibits cell death (7, 8, 9). Glucocorticoids, which are important antiinflammatory drugs in the treatment of asthma, markedly inhibit TNF-α-induced NF-κB activation in HeLa, Jurkat, and 2B4 cells (10, 11, 12, 13, 14). One mechanism by which glucocorticoids inhibit NF-κB activation is by increasing transcription of IκBα gene expression. IκBα is an inhibitory protein that binds and inactivates NF-κB. In some cell types, glucocorticoids are ineffective in abrogating cytokine-induced NF-κB activation (15, 16). These studies suggest that the role of NF-κB in cytokine signaling is unique to the cell and tissue studied.
NF-κB is implicated as a pivotal transcription factor mediating chronic inflammatory responses in asthma, rheumatoid arthritis, psoriasis, and inflammatory bowel disease (17). Activation of NF-κB stimulates recruitment and activation of T and B lymphocytes, eosinophils, and macrophages, which amplify and perpetuate the inflammatory lesion. Despite considerable interest in studying the role of NF-κB in regulating immunocyte function, few investigators have studied the role of NF-κB in regulating functional processes in structural cells such as smooth muscle and fibroblasts. In a variety of diseases, smooth muscle cells can play an important immunomodulatory role. Although TNF-α and IL-1β mediate some effects, in part, through activation of NF-κB, other transcription factors likely play critical roles in transducing cytokine effects especially in nonhemapoietic cell types. In cultured vascular smooth muscle cells, the role of NF-κB activation in mediating growth remains controversial. Studies suggest that NF-κB may or may not modulate thrombin- and platelet-derived growth factor-induced vascular smooth muscle cell proliferation (1, 2). NF-κB-dependent pathways also regulate oxidant-induced increases in A1 adenosine receptor expression and TNF-α-induced expression of the bradykinin (B1) receptor in vascular smooth muscle cells. In human airway smooth muscle (ASM) cells, the role as well as the activation of NF-κB in cytokine signaling has not been investigated. Only one study from our laboratory showed that cross-linking CD40, a member of the TNF-α receptor family, activates NF-κB (18). In addition, a variety of proinflammatory genes activated by TNF-α and IL-1β in human ASM cells contain NF-κB binding sites in their promoter region (reviewed in Ref. 17). NF-κB appears to play a central role in mediating cytokine-induced synthetic functions in human ASM cells.
In this study, we demonstrate that cytokine-mediated ICAM-1 expression, in contrast to cyclooxygenase (COX)-2 expression, was not suppressed by dexamethasone. In addition, dexamethasone had no effect on NF-κB activation following stimulation with TNF-α and IL-1β. Our data show that the inability of dexamethasone to affect synthesis of ICAM-1 may be due, in part, to the cytokine activation of a steroid-insensitive NF-κB pathway.
Materials and Methods
Materials
Dexamethasone was purchased from Sigma (St. Louis, MO). HAM/F12, FCS, trypsin, and antibiotics (penicillin and streptomycin) were obtained from Life Technologies (Grand Island, NY). IL-1β and TNF-α were purchased from Boehringer Mannheim (Minneapolis, MN).
Smooth muscle cell culture and characterization
The culture of human ASM cells was performed as described elsewhere (19, 20). The characterization of the cultured cells as smooth muscle cells was confirmed by immunostaining with anti-smooth muscle α-actin as previously described (19).
EMSA
Nuclear extracts were prepared as described previously (21). Briefly, cells grown in serum-free medium were preincubated with or without dexamethasone and stimulated with TNF-α for 1 h at 37°C. The cells were then washed with ice-cold PBS before they were resuspended in ice-cold lysis buffer (10 mM Tris-HCl, pH 8, 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, and 2 μg/ml leupeptin and aprotinin) for 15 min. Nuclei were separated from the cytoplasm by centrifugation at 4°C for 5 min, washed, and resuspended in nuclear extraction buffer (20 mM Tris-HCl, pH 8, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol) for 10 min on ice. The resulting nuclear proteins (5–10 μg) were incubated with 3 μg of poly(dI-dC) and 0.4 pmol of double-stranded 32P-labeled oligonucleotide containing the NF-κB consensus binding site (5′-AGTTGAGCGGACTTTCCCAGGC-3′; Santa Cruz Biotechnology, Santa Cruz, CA) in a total volume of 20 μl in a buffer containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol. After 30 min at room temperature, samples were separated on a nonreducing 4% polyacrylamide gel using Tris running buffer (1 M, pH 7.5) containing 3 M sodium acetate and 0.5 M EDTA. The gels were dried and autoradiographed with intensifying screens at −70°C.
Western blot analysis
Western blotting for IκBα and COX-2 were performed as described previously (21, 22). Briefly, ASM cells were washed with cold PBS and resuspended in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, and 10 μg/ml aprotinin and leupeptin. The cell lysate was kept on ice for 20 min and centrifugated at 12,000 rpm for 5 min. The cytoplasmic proteins (30 μg) were separated by SDS-PAGE on a 12.5% gel for IκBα and an 8% gel for COX-2 proteins. Proteins were transferred onto nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The membrane was then blocked for 1 h with 3% BSA in TBS and incubated for 2 h with either rabbit polyclonal IgG anti-IκBα Ab (Santa Cruz Biotechnology) or mouse monoclonal IgG anti-COX-2 Ab (Cayman Chemical, Ann Arbor, MI). The appropriate secondary HRP-conjugated Ab (Boehringer Mannheim, Minneapolis, MN) was added and complexes detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and autoradiographed.
Flow cytometry analysis
Flow cytometry was performed as described previously with slight modifications (23
Transfection of human ASM cells
Transfections of human ASM cells were performed as described previously (24). Briefly, ASM cells were transfected with 2 μg of pNF-κB-Luc designed for monitoring the NF-κB signal transduction pathway (Clontech, Palo Alto, CA) and 2 μg of pSV-β-galactosidase control vector to normalize transfection efficiencies (Promega, Madison, WI). Eighteen hours after transfection, the cells were rendered quiescent in medium containing 0.2% FBS for 16 h and exposed to TNF-α or IL-1β for 4 h. Cells were then harvested and luciferase and β-galactosidase activities were assessed as directed by Promega.
Statistical analysis
All data are expressed as means ± SEM. Comparisons between treatment were made using Student’s t test for paired values. The mean values were considered significantly different when the probability of the event was below 5%.
Results
Dexamethasone does not suppress cytokine-mediated ICAM-1 expression
To examine critical signaling events that modulate cytokine-mediated ICAM-1 expression, we examined whether glucocorticoids inhibit cytokine-induced ICAM-1 expression in human ASM cells. Previously, we showed that TNF-α and IL-1β induce expression of ICAM-1 on human ASM (23, 25). TNF-α induced a 7.1 ± 1-fold increase in ICAM-1 expression after 4 h that was reduced to 3.7 ± 0.1 and 3.6 ± 0.2 in the presence of 1 or 10 μM dexamethasone, respectively (Fig. 1⇓A). IL-1β induced a 5.3 ± 0.6-fold increase in ICAM-1 expression that was reduced to 3.14 ± 0.2 and 2.9 ± 0.2 in the presence of 1 or 10 μM dexamethasone, respectively (Fig. 1⇓A). In contrast, when cells were exposed to cytokines for 24 h, dexamethasone failed to suppress the expression of ICAM-1 (Fig. 1⇓B). Dexamethasone did not affect and even potentiated TNF-α-stimulated ICAM-1 expression after 24 h. The net increases in mean fluorescence over background were 17.1 ± 0.6 (n = 4, p < 0.01), 17.3 ± 1.3 (n = 4, NS), and 18.7 ± 0.7 (n = 4, p < 0.05) in cells treated with TNF-α alone and in the presence of dexamethasone 0.01 μM and 1 μM, respectively. In contrast, IL-1β-mediated ICAM-1 expression after 24 h was partially inhibited by dexamethasone with net increases in mean fluorescence over background being 6.5 ± 0.6 (n = 4, p < 0.01 vs basal), 6 ± 0.7 (n = 4, NS), and 5.4 ± 0.58 (n = 4, p < 0.05) in cells treated with IL-1β alone and in the presence of dexamethasone 0.01 μM and 1 μM, respectively (Fig. 1⇓B). Taken together, these data clearly suggest that cytokine-mediated ICAM-1 induction at early time points is partially sensitive to glucocorticoids, while at later time points glucocorticoids have little effect.
Effect of dexamethasone on early and late expression of ICAM-1 induced by TNF-α and IL-1β. ASM cells were pretreated with diluent or dexamethasone at the indicated concentrations for 1 h before stimulation with 10 ng/ml TNF-α or 1 ng/ml IL-1β for 4 h (A) or 24 h (B). FACS analysis of ICAM-1 expression was assessed as described in Materials and Methods. Values shown are mean ± SEM, n = 4; #, p < 0.05 compared with cells treated with diluent, and ∗∗, p < 0.01 or ∗, p < 0.05 compared with cells with cytokines alone.
Dexamethasone inhibits IL-1β-mediated COX-2 expression
Previous studies in human ASM cells have shown that the rapid COX-2 expression induced by IL-1β is suppressed by dexamethasone (22, 26). To ascertain whether concentrations of dexamethasone used in our experimental conditions were effective, the inducible expression of COX-2 proteins by IL-1β was examined after pretreating cells with 1 μM dexamethasone. After 6 h of incubation with IL-1β, there was a dramatic increase in the expression of COX-2 by human ASM cells (Fig. 2⇓). In contrast, TNF-α had no effect on COX-2 expression (data not shown). This effect is completely abrogated by pretreatment of the cells with dexamethasone. Reprobing the same blot with anti-COX-1 Ab revealed that COX-1 protein remained unchanged in the three different conditions (Fig. 2⇓). These data suggest that 1 μM dexamethasone completely inhibits COX-2 expression but has no effect on the induction of ICAM-1 by cytokines.
Dexamethasone suppresses induction of COX-2 by IL-1β. ASM cells were preincubated for 1 h with 1 μM dexamethasone and exposed to 1 ng/ml IL-1β for 6 h. Cells were lysed, and cytoplasmic extracts were prepared and assayed for COX-2 or COX-1 by immunoblot analysis. Results are representative of two different experiments.
TNF-α and IL-1β induce NF-κB activation in human ASM
To date, no studies have described the activation of transcription factors by cytokines in human ASM cells. To study the effect of cytokines on NF-κB activation, ASM cells were transfected with a construct consisting of κB enhancer elements and a luciferase reporter. Transfected cells were stimulated with cytokines and luciferase activity in cell lysates was measured. Treatment of transfected cells with TNF-α or IL-1β for 4 h induced a 3.4 ± 0.5- and 7.6 ± 1.6-fold increase in luciferase activity, respectively, as compared with unstimulated cells (Fig. 3⇓C). In contrast, neither bradykinin nor thrombin, agonists that activate G-protein-coupled receptors and mobilize calcium (20, 27), had any effect on luciferase activity (Fig. 3⇓C).
Cytokines induce NF-κB activation by promoting IκBα degradation. ASM cells were stimulated for the indicated time with cytokine, and cytoplasmic extracts were prepared and assayed for IκBα by Western blot analysis. The time course of IκBα degradation is shown following stimulation with either 10 ng/ml TNF-α (A) or 1 ng/ml IL-1β (B). ASM cells were also preincubated with 10 μM cycloheximide (CHX) for 1 h and then stimulated with 10 ng/ml TNF-α or 1 ng/ml IL-1β for 1 h and then assayed for IκBα. C, IL-1β and TNF-α but not bradykinin or thrombin activate κB-luciferase reporter expression in human ASM cells. Cells were transfected with 2 μg of κB-luciferase reporter construct, placed in media containing 0.2% serum for 16 h, and then stimulated with 10 ng/ml TNF-α, 1 ng/ml IL-1β, 1 μM bradykinin (Bk), or 1 U/ml thrombin (Thr) for 4 h. Luciferase activity in cell extracts was normalized for β-gal activity as described in Materials and Methods. Results are representative of three different experiments.
NF-κB activation is known to be a direct result of IκBα degradation, its cytosolic inhibitor (3). Western blot analysis of IκBα protein in cytoplasmic extracts of treated cells demonstrated that TNF-α (Fig. 3⇑A) or IL-1β (Fig. 3⇑B) induced IκBα degradation in a time-dependent manner, which was almost complete by 15 min, with reappearance of IκBα after 60 min. The resynthesis of IκBα was shown to be dependent upon protein synthesis, because pretreatment of cells with cycloheximide completely prevented the reappearance of IκBα after 60 min (Fig. 3⇑B), without affecting IκBα degradation (data not shown). These data suggest a potential role of NF-κB in cytokine-induced cellular responses in ASM cells.
Dexamethasone does not affect TNF-α-mediated IκBα degradation, NF-κB translocation, or NF-κB-mediated transcription
In some cell types, dexamethasone inhibits cytokine-induced NF-κB activation. Therefore, we examined the ability of dexamethasone to inhibit cytokine-mediated NF-κB activation and IκBα degradation. Pretreatment of ASM cells with dexamethasone had no effect on TNF-α- or IL-1β-mediated IκBα degradation (Fig. 4⇓A) or IκBα resynthesis (Fig. 4⇓B).
Dexamethasone does not prevent cytokine-mediated IκBα degradation or IκBα resynthesis. Human ASM cells pretreated with 1 μM dexamethasone (Dex) for 1 h were stimulated with 10 ng/ml TNF-α or 1 ng/ml IL-1β for 15 min (A) or 60 min (B). Cytoplasmic extracts were prepared and assayed for IκBα by Western blot analysis as described in Materials and Methods. Results are representative of three different experiments.
Using EMSA, we found that pretreatment of ASM cells with dexamethasone had no effect on TNF-α-mediated NF-κB activation (Fig. 5⇓A). In transfected cells with an NF-κB-luciferase reporter construct, we found that TNF-α induced a 4-fold increase in luciferase activity (Fig. 5⇓B). Pretreatment of the cells with dexamethasone had no effect on this response (Fig. 5⇓B). Taken together, these results demonstrate that NF-κB activation by cytokines in human ASM cells is unaffected by concentrations of dexamethasone known to suppress NF-κB in other cell type (28).
Dexamethasone does not prevent cytokine-mediated NF-κB DNA binding or NF-κB-mediated reporter activity. A, Human ASM cells were pretreated with 1 μM dexamethasone (Dex) for the indicated times and stimulated with 10 ng/ml TNF-α for 60 min. Nuclear extracts were prepared and assayed for NF-κB by EMSA as described in Materials and Methods. Results are representative of two different experiments. B, ASM cells were transfected with 2 μg κB-luciferase reporter construct. Cells were pretreated with 1 μM dexamethasone (Dex) for 1 h, then stimulated with TNF-α (10 ng/ml) for 4 h, where indicated. Luciferase activity was compared with untreated (basal) cells. Results are representative of three experiments.
NF-κB is necessary for cytokine-stimulated ICAM-1 but not COX-2 protein expression
Because functional NF-κB regulatory elements are present within the promoter region of the ICAM-1 gene (29, 30), we hypothesized that NF-κB activation is necessary for cytokine-mediated effects on ICAM-1 expression in human ASM cells. We showed that IκBα degradation is an obligatory step in the activation of NF-κB. To block NF-κB activation, cells were pretreated with protease inhibitors N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N-acetyl-l-leucinyl-l-leucinyl-norleucinal (ALLN), which have been shown to prevent IκBα degradation and NF-κB translocation into the nucleus (reviewed in Ref. 4). The effect on ICAM-1 expression was examined. Pretreatment of cells with TPCK (25 μM) or ALLN (50 μM) for 1 h abrogated both cytokine-mediated IκBα degradation (Fig. 6⇓, A and B) and to a similar extent the expression of ICAM-1. TPCK was found to be more potent than ALLN in preventing IκBα degradation (Fig. 7⇓). In contrast, we observed that induction of COX-2 expression by IL-1β was not affected by the same concentration of TPCK (data not shown).
Inhibition of cytokine-mediated IκBα degradation by TPCK or ALLN. A, Cells were preincubated for 1 h with 25 μM TPCK or 50 μM ALLN and then stimulated with 10 ng/ml TNF-α or 1 ng/ml IL-1β for 4 h. IκBα was assessed as described in Materials and Methods. B, Densitometric quantitation of IκBα bands shown in A (expressed as percentage of basal). Data are representative of three different experiments.
Inhibition of IκBα degradation prevents cytokine mediated up-regulation of ICAM-1 expression. Cells were preincubated for 1 h with 25 μM TPCK or 50 μM ALLN, then stimulated with 10 ng/ml TNF-α or 1 ng/ml IL-1β for 4 h. Expression of ICAM-1 was assessed by flow cytometry. Values of ICAM-1 fluorescence are shown as mean ± SEM, n = 4; ∗∗, p < 0.05 compared with cells treated with cytokines alone; #, p < 0.01 compared with untreated cells.
Discussion
Proinflammatory mediators found in the airways of patients with asthma play an important role in promoting cellular recruitment as well as in inducing smooth muscle cell hyperreactivity. Inflammatory cytokines, such as IL-1β and TNF-α, that do not by themselves induce ASM contraction can prime ASM cells to become hyperresponsive to directly acting bronchoconstrictors (reviewed in Ref. 31). In addition, these same cytokines can up-regulate smooth muscle cell expression of the cell adhesion molecules ICAM-1 and VCAM-1, which can support adhesion of activated leukocytes (23, 25). In this study, we have characterized a potentially important signal transduction pathway in ASM cells, which involves a glucocorticoid-insensitive activation of NF-κB by TNF-α and IL-1β. Activation of NF-κB is necessary for expression of ICAM-1, but not for the expression of COX-2. In addition, dexamethasone completely inhibited cytokine-induced COX-2 expression but not that of ICAM-1. These data provide further evidence that IL-1β and TNF-α induce activation of multiple signaling pathways for the induction of gene expression.
Our initial studies focused on the ability of glucocorticoids to inhibit TNF-α-induced ICAM-1 expression in ASM cells. We found that at early time points glucocorticoids partially inhibited expression. The mechanisms by which steroids affect early ICAM-1 expression induced by cytokines are not delineated in the present study; however, several hypotheses could be considered. In addition to NF-κB binding sites, the promoter region of the ICAM-1 gene contains various putative transcriptional regulatory elements such as AP-1 and AP-1/Ets, AP-3, and CCAAT/enhancer-binding protein (C/EBP) (29, 30). The use of reporter plasmids and their 5′ deletion derivatives demonstrated that NF-κB-dependent gene transcription is a rather complex phenomenon and might involve a synergistic action of NF-κB with other transcription factors (32, 33, 34). Therefore, it is possible that dexamethasone could suppress ICAM-1 expression at an early time point by interfering with other transcription factors such as C/EBP and AP-1 that have been shown to be involved in the transcriptional regulation of the ICAM-1 gene. Support for this hypothesis comes from investigators showing that dexamethasone, while having a modest effect on NF-κB activation, completely suppressed the activation of AP-1 in response to IL-1β (33). More experiments are needed to determine the precise role of transcription factors involved in ICAM-1 expression by cytokines to characterize the effects of dexamethasone inhibition of TNF-α-induced ICAM-1 expression. Interestingly, the modest inhibitory effect of dexamethasone on ICAM-1 expression was not sustained over 24 h. Similar findings have been described in human hepatocytes (35), human fibroblasts (36), and in vascular endothelium (37, 38), where cytokine-induced ICAM-1 expression was either unchanged or only partially decreased by steroids. In contrast, we and others have shown that dexamethasone dramatically inhibits cytokine-induced COX-2 expression in ASM cells (22, 26, 39). More recently, two studies performed in human ASM cells reported that the expression and release of chemokines IL-8 or RANTES in response to TNF-α were partially inhibited by dexamethasone (40, 41). This suggests that both steroid-sensitive and -insensitive pathways are activated by cytokines and modulate gene expression in ASM cells.
It is believed that many of the antiinflammatory effects of glucocorticoids are due to their interference with transcription factors, such as NF-κB or AP-1 (reviewed in Ref. 17). Therefore, we sought to determine the mechanisms underlying this steroid-insensitive signaling pathway by studying the effects of glucocorticoids on IL-1β- and TNF-α-induced NF-κB activation. We found that neither NF-κB DNA binding nor NF-κB-mediated luciferase activity was inhibited by dexamethasone pretreatment. This lack of inhibition was seen even when the cells were preincubated with dexamethasone for as long as 24 h. Further studies demonstrated that both the time course of IκBα degradation and resynthesis were unaffected by dexamethasone. The functional significance of IκBα degradation in mediating cytokine-induced NF-κB activation in human ASM cells was examined by using the inhibitors TPCK and ALLN, which are known to suppress NF-κB activation by blocking IκBα degradation (reviewed in Ref. 4). We found that pretreatment with TPCK and ALLN inhibited cytokine-mediated IκBα degradation and abrogated ICAM-1 expression in human ASM cells, without inducing changes in cellular viability. This result is not entirely surprising because NF-κB regulatory elements are present within the promoter region of the ICAM-1 gene, and studies have shown that NF-κB activation is critical for the regulation of ICAM-1 gene transcription (29, 30, 32, 36). Notably, however, TPCK and ALLN had no effect on cytokine-induced COX-2 expression.
We found that not only is IL-1β approximately two times more potent than TNF-α in activating NF-κB reporter activity, but also that IL-1β, but not TNF-α, stimulated COX-2 protein in human ASM cells. Similarly, we have demonstrated that bradykinin, a calcium mobilizing agent (20, 27), which can induce COX-2 expression in human ASM cells (22), does not activate NF-κB in human ASM cells. In other cell types, IL-1β induces COX-2 gene expression that was associated with DNA-binding activity of AP-1, NF-κB, and IFN-γ activation site in keratinocytes (33). In mouse osteoblastic cells, COX-2 induction involved NF-κB activation but was found to be ∼80% regulated by the transcription factor NF-IL-6 (34). These data suggest that NF-κB activation is neither necessary nor sufficient to induce COX-2 expression in ASM cells. In contrast, NF-κB activation appears to be necessary for both TNF-α- and IL-1β-induced expression of ICAM-1. Together, our results therefore demonstrate that the signal transduction pathways mediating cytokine-induced ICAM-1 expression in human ASM cells are different from those used to stimulate COX-2 expression, and this may explain their differential sensitivity to steroids.
Some reports describe that glucocorticoids act by direct protein-protein interaction between the steroid receptor and NF-κB (13) or by the induction of IκBα protein (14, 28, 42). In contrast, others have demonstrated that steroids modulate gene transcription through direct binding to glucocorticoid responsive elements that regulate either the activation or repression of gene transcription. Because the ICAM-1 gene lacks glucocorticoid responsive elements in the promoter region (29, 30), it is likely that steroids affect ICAM-1 through cross-coupling mechanisms with NF-κB or through the induction of IκBα. However, our data demonstrate that glucocorticoids have no effect on either NF-κB DNA-binding activity or on IκB synthesis, nor do they inhibit cytokine-induced ICAM-1 expression. In contrast, COX-2 expression is markedly diminished by glucocorticoids, which may represent the effects on other transcription factors, such as AP-1, IFN-γ activation site, or NF-IL-6, because the COX-2 promoter does not have a glucocorticoid responsive element site (33). Similarly, several studies have demonstrated that TNF-α induces ICAM-1 transcription through C/EBP and NF-κB elements within the ICAM-1 promoter in endothelial cells (32). TNF-α-mediated optimal activation of VCAM-1 promoter requires both Sp1 and NF-κB (43). These data suggest that at early time points, cytokines stimulate the differential activation of transcription factors that may lead to different patterns of gene transcription, such as ICAM-1 and COX-2 genes, and that may express different sensitivity to the suppressive action of steroids. Recently, investigators described that posttranscriptional mechanisms involving a repression of COX-2 mRNA played an important role in inhibiting IL-1β-mediated COX-2 expression (44). These findings suggest potential mechanisms, which are unrelated to the modulation of gene transcription, can account for the effect of dexamethasone on IL-1β-induced COX-2 expression in human ASM cells. However, additional experiments are needed to identify the precise signaling events that mediate these effects.
After prolonged exposure of cells with cytokines, steroid effects on ICAM-1 expression are more complex. Both TNF-α and IL-1β have been shown to release a variety of proinflammatory mediators, such as RANTES, IL-6, and IL-8, which are not completely blocked by dexamethasone (40, 41). In our laboratory, we found that TNF-α-induced RANTES expression was completely abrogated by dexamethasone (our unpublished observations). Therefore, it is plausible that the secreted proinflammatory agents could reduce steroid action either by potentiating cytokine effects, as observed in this study for TNF-α-induced ICAM-1 expression, or by impairing steroid responsiveness. One recent study described that the release of NO in mouse fibroblasts altered steroid responsiveness by decreasing both steroid receptor affinity and number (45). Synthesis of NO by human ASM cells has not been described, although a recent report describes the ability of LPS to increase NO synthase mRNA in rat ASM cells (46). Because glucocorticoids did not suppress cytokine-induced ICAM-1 expression, it seems unlikely that steroid modulation of the NO pathway plays a role in cytokine-induced ICAM-1 expression.
Modification of ASM function by cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma (reviewed in Ref. 31). Proinflammatory cytokines up-regulate ASM expression of ICAM-1 and VCAM-1, which promotes lymphocyte adhesion and induces smooth muscle cell DNA synthesis (23). Increased levels of ICAM-1 have been detected in asthmatic patients after allergen exposure (47, 48) or during asthma attacks (49). mAbs against ICAM-1 are able to inhibit both inflammatory cell infiltrate and acquired bronchial hyperresponsiveness (50). In addition, a recent study demonstrates the activation of NF-κB DNA-binding activity in bronchial biopsies obtained from patients with asthma (51). Based on our data and those of others (40, 41), the inability of steroids to inhibit cytokine-induced synthetic responses in ASM cells, involving the NF-κB pathway, may have important consequences for the local inflammatory response in airways. Our data support the notion that the antiinflammatory effects of corticosteroids in asthma may not be due to modulation of cytokine-induced NF-κB activation or ICAM-1 expression in human ASM cells. Collectively, these studies open a new and important area of investigation, delineating cytokine-mediated signal transduction pathways that are insensitive to steroids.
Acknowledgments
We thank Dr. Raymond Penn for providing the adenovirus Ad5-GPT used for transfection studies and Mary McNichol for assistance in the preparation of the manuscript.
Footnotes
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↵1 This work was supported by National Institutes of Health Grant HL03202 and the McCabe Fund of the University of Pennsylvania (to A.L.L.) and Grants HL55301 and AI40203, and an American Lung Association Career Investigator Award (to R.A.P.).
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↵2 Address correspondence and reprint requests to Dr. Yassine Amrani, Pulmonary, Allergy, and Critical Care Division, Room 808 East Gates Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail address: amrani{at}mail.med.upenn.edu
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↵3 Abbreviations used in this paper: IκB, NF-κB inhibitor; ASM, airway smooth muscle; COX, cyclooxygenase; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; ALLN, N-acetyl-l-leucinyl-l-leucinyl-norleucinal.
- Received April 6, 1999.
- Accepted May 28, 1999.
- Copyright © 1999 by The American Association of Immunologists
References
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