help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on June 29, 2006, doi:10.1165/rcmb.2005-0407OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0407OCv1
35/6/697    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thompson, C.
Right arrow Articles by Rola-Pleszczynski, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Thompson, C.
Right arrow Articles by Rola-Pleszczynski, M.
American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 697-704, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0407OC

CysLT1 Receptor Engagement Induces Activator Protein-1– and NF-{kappa}B–Dependent IL-8 Expression

Charles Thompson, Alexandre Cloutier, Ynuk Bossé, Maryse Thivierge, Christian Le Gouill, Pierre Larivée, Patrick P. McDonald, Jana Stankova and Marek Rola-Pleszczynski

Immunology Division, Department of Pediatrics, and Pulmonary Division, Department of Medicine, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada

Correspondence and requests for reprints should be addressed to Marek Rola-Pleszczynski, Immunology Division, Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, 3001 North 12th Avenue, Sherbrooke, PQ, J1H 5N4 Canada. E-mail: marek.rola-pleszczynski{at}usherbrooke.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Because cysteinyl-leukotrienes (cysLTs) are major protagonists in the pathophysiology of human asthma, and because neutrophils are involved in the more severe form of asthma, we studied the potential for leukotriene (LT) D4 to induce synthesis of the chemokine IL-8 through activation of the CysLT1 receptor. We found LTD4 to induce IL-8 gene expression in monocytic THP-1 cells and human dendritic cells with complete abrogation by selective CysLT1 antagonists. Human embryonic kidney–293 cells stably transfected with CysLT1 were used to better study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with graded concentrations of LTD4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-{kappa}B, activator protein (AP)–1, and NF–IL-6 binding elements revealed a requirement for NF-{kappa}B and AP–1, but not NF–IL-6, in LTD4-induced activation of the IL-8 promoter. Overexpression of dominant-negative I{kappa}B{alpha} inhibited the IL-8 transactivation induced by LTD4. NF-{kappa}B DNA binding activity was induced by LTD4, as determined by electrophoretic mobility shift assays, and could be supershifted by antibodies against p50 and p65. Supershift assays after LTD4 stimulation also indicated the formation of a c-Jun/c-Fos complex. Moreover, our results demonstrate that LTD4 upregulates the expression of c-fos and c-jun at the mRNA level. Our data show for the first time that LTD4, via the CysLT1 receptor, can transcriptionally activate IL-8 production, with involvement of the transcription factors p50, p65, Fos, and Jun. These findings provide mechanistic and potentially therapeutic elements for modulation of the inflammatory component of asthma.

Key Words: activator protein–1 • chemokines • cysteinyl-leukotrienes • inflammation • NF-{kappa}B • signaling



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study demonstrates that LTD4 can transcriptionally induce the production of the chemokine IL-8, a potent chemoattractant for neutrophils and other leukocytes, which may be relevant for the pathogenesis of COPD, and occupational and severe asthma.

 
Cysteinyl-leukotrienes (cysLTs), leukotriene (LT) C4, LTD4, and LTE4 are potent lipid mediators involved in the pathogenesis of asthma and the resulting pulmonary inflammation. They are mainly released by eosinophils, mast cells, and macrophages in the airways (1), and increased levels of LTs have been found in the bronchoalveolar lavage (BALF) (2, 3) and sputum (4, 5) of patients with asthma. CysLTs act on at least two G-protein–coupled receptors, CysLT1 and CysLT2 (6, 7). High expression of CysLT1 has been demonstrated in the lung, spleen, and peripheral blood leukocytes (6, 8). CysLT1 seems to be responsible for the acute bronchoconstriction effects and mucus hypersecretion induced by cysLTs (9–11). Some evidence also suggests that cysLTs act as chemotactic agents for eosinophils (12). In this regard, the suggested proinflammatory role of cysLTs has been reinforced by the observation that selective CysLT1 antagonists reduce the amount of eosinophil infiltration in the airways (13, 14).

IL-8, a potent mediator of inflammation, is the prototypical member of the CXC chemokine family. Expression of IL-8 is elevated in BALF and sputum of patients with asthma (15, 16). In addition, IL-8 is a powerful activator, chemoattractant, and survival agent for polymorphonuclear leukocytes (17). Neutrophils are the predominant inflammatory cells in the airways of patients with acute severe asthma (15, 16) and professional asthma (18). Furthermore, chronic obstructive pulmonary disease (COPD), which is characterized by abnormal inflammatory responses of the airways and lung parenchyma, is associated with airway neutrophilia (19, 20). Airways of patients with COPD also contain high levels of IL-8 (20). In addition to neutrophils, IL-8 also activates different subsets of T cells, as well as natural killer cells and B cells (21). IL-8 gene expression is mainly regulated by three elements: activator protein (AP)-1, NF-{kappa}B, and NF–IL-6, all present in the IL-8 gene promoter (22). NF-{kappa}B is a homo- or heterodimer composed of different members of the Rel family of proteins: NF-{kappa}B1 (p50), NF-{kappa}B2 (p52), c-Rel, RelA (p65), and RelB (23, 24). The most abundant form of NF-{kappa}B heterodimers is composed of p50 and p65 subunits (25). On the other hand, AP-1 is a dimeric transcription factor that includes members of the Jun and Fos families. Jun family proteins, represented by c-Jun, JunB, and JunD, and Fos family proteins, represented by c-Fos, FosB, Fra-1, and Fra-2, form homo- or heterodimers that bind to the DNA consensus sequence TGAC/GTCA and regulate the activation of different target genes (26, 27). NF-{kappa}B and AP-1 transcription factors play a pivotal role in the regulation of multiple genes involved in immune and inflammatory responses (25).

Asthma is characterized by chronic inflammation, with infiltration of different leukocytes and high expression of numerous inflammatory proteins in the airways. In severe asthma, both higher levels of cysLTs (28, 29) and increased numbers of neutrophils have been reported (15, 16). We thus hypothesized that LTD4 could modulate the expression of a chemokine, such as IL-8. This would also provide us with the opportunity to study the possible signaling pathways driven by CysLT1. In the present report, we demonstrate that LTD4 could upregulate the expression of IL-8 in monocytic cells. Moreover, human embryonic kidney (HEK)–293 cells stably transfected with CysLT1 also showed modulation of IL-8 production by LTD4 and served as a model to study CysLT1 signaling. Our results show that modulation of IL-8 expression through the CysLT1 receptor was transcriptional and involved the NF-{kappa}B and AP-1 signaling pathways.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Antibodies and Reagents
Specific antibodies (Abs) against p-I{kappa}B{alpha} Ser 32(9241S) and p-p65 Ser 536(3031S) were purchased from Cell Signaling Technology (Beverly, MA) and I{kappa}B{alpha} (sc-3718), p65 (sc-8008X), p50 (sc-7178X), c-Jun (sc-1694X), c-Fos (sc-52X), JunD (sc-74X), JunB (sc-46X), FosB (sc-48X), CREB1 (sc-240X), and CREB2 (sc-200X) were from Santa Cruz Biotechnology (Santa Cruz, CA). LTD4 was obtained from Cayman Chemical (Ann Arbor, MI). Aprotinin, 4-(2 aminoethyl)benzenesulfonyl fluoride, leupeptin, NaF, soybean trypsin inhibitor, and Na3VO4 were from Sigma-Aldrich (Oakville, ON, Canada). Hygromycin B (Wisent, St.-Bruno, PQ, Canada) and FuGENE6 were purchased from Roche Diagnostics Canada (Laval, PQ, Canada). Montelukast (MK-476) was obtained from Merck Frosst (Pointe-Claire, PQ, Canada), and Zafirlukast from AstraZeneca (Lund, Sweden).

Plasmids
pCMVintron-cmyc-CysLT1R IRES-hygro, a bicistronic transcript coding for cmyc-CysLT1R and hygromycin resistance, stabilized by a 5' beta-globin intron and under the control of a CMV promoter, was constructed in our laboratory. The dominant negative form of I{kappa}B{alpha} mutated at serines 32 and 36 were generously provided by Dr. Christian Jobin (University of North Carolina at Chapel Hill). pTA-Luc, pNF-{kappa}B-TA-Luc, and pAP-1-TA-Luc were from Clontech (BD Biosciences, Mississauga, ON, Canada). The human IL-8 promoter luciferase constructs IL-8 WT, IL-8{Delta}AP-1, IL-8{Delta}NF-{kappa}B, and IL-8{Delta}NF–IL-6 were generously provided by Dr. Allan R. Brasier from the University of Texas Medical Branch (30).

Cells
Immature primary dendritic cells were derived from human peripheral blood mononuclear leukocytes and generated as previously described (31). Human monocytic THP-1 cells (American Type Culture Collection [ATCC], Rockville, MD) were cultured in RPMI-1640 medium (Life technologies, Burlington, ON, Canada) supplemented with 10% FBS, 100U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich) HEK293 cells (ATCC) were grown in Dulbecco's modified Eagle's medium with glucose (Life technologies), supplemented with 10% FBS (Sigma-Aldrich), and gentamicin sulfate (40 µg/ml).Transient transfections were performed with FuGENE 6, and experiments were performed 48 h after transfection.

HEK293 Stably Expressing CysLT1
HEK293 cells grown at 50% confluence in 60-mm Petri dishes were stably transfected with 4 µg of pCMVintron-cmyc-CysLT1R using 10 µl of FuGENE 6. At 48 h after transfections, hygromycin B was added at a final concentration of 300 µg/ml. Cells were then cultured for 2 wk in medium containing hygromycin B and isolated for clonal selection. Cells clones were analyzed for Myc expression by FACScan flow cytometer (BD Bioscience, San Jose, CA). Positive clones were maintained in 100 µg/ml hygromycin B. These cells are referred to as 293LT1.

Northern Blot Analysis
Total cellular RNA was extracted using TriPure according to the manufacturer's instructions (Roche Diagnostics Canada), 15 µg of total RNA was separated by electrophoresis on 1% agarose, and the material was transferred onto a Hybond N+ (Amersham Pharmacia Biotech, Baie d'Urfé, PQ, Canada) membrane for Northern blot analysis. Human IL-8 cDNA probe (0.5 kb EcoRI fragment) and human c-Fos cDNA probe were obtained from ATCC; human c-Jun probe was kindly provided by Dr. Claude Asselin, Université de Sherbrooke. 28S or 18S were used as internal controls. The probes were labeled with Ready-To-Go DNA labeling beads (dCTP) (Amersham Biosciences, Piscataway, NJ) using [{alpha}-32P] dCTP (specific activity, 3,000 Ci/mmol; Amersham Pharmacia Biotech). Membranes were prehybridized for 4 h in a mixture containing 120 mM Tris (pH 7.4), 600 mM NaCl, 8 mM EDTA (pH 8), 0.1% sodium pyrophosphate, 0.2% SDS, and 100 g/ml heparin; hybridization was performed overnight at 71°C (IL-8) or 68°C (c-Fos and c-Jun) in the same mixture, in which the concentration of heparin was increased to 625 µg/ml and 10% dextran sulfate was added. The membranes were then washed once at room temperature for 20 min in 2x SSC (1x SSC: 0.15 M NaCl, 0.15 M sodium citrate [pH 7]) and once with 0.1x SSC. The membranes were exposed to Hyperfilm MP (Amersham Pharmacia Biotech) with intensifying screens at –80°C.

Western Blot Analysis
293LT1 cells in 6-well plates were incubated in medium without serum for 24 h, stimulated with LTD4 (10 nM) for the indicated times, and lysed in buffer (50 mM Tris [pH 7.5], 1 mM EGTA, 150 mM NaCl, 1 mM NaF, 1 mM Na3VO4, 1% TRITON X-100, 0.25% sodium deoxycholate, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 10 µg/ml soybean trypsin inhibitor) for 30 min on ice. Total lysates were separated on 10% SDS-PAGE, and transferred to Trans-Blot nitrocellulose membrane (BioRad Laboratory, Inc., Hercules, CA). Membranes were blocked with TBS with 5% dry milk for 1 h and incubated with specific Abs in TBS/0.1% Tween and 5% dry milk overnight at 4°C. After washing and incubating with secondary Abs, an ECL detection system was used for protein detection (Amersham Pharmacia Biotech). Membranes were stripped by incubation in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 10 mM 2-ME for 30 min at 50°C. After washing, membranes were reprobed with the appropriate Abs and developed as described previously here.

Electrophoretic Mobility Shift Assays
293LT1 cells were cultured in 6-well plates until nearly confluent; cells were starved overnight. Cells were then stimulated with LTD4 or EtOH for indicated times, and incubations were stopped by adding an equal volume of ice-cold PBS containing 10 mM NaF and 1 mM Na3VO4. Cells were collected by gentle scraping and centrifuged at 1,000 x g for 3 min at 4°C. The resulting cell pellets were resuspended in ice-cold lysis buffer (10 mM HEPES [pH 7.90], 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM EGTA, and 0.5 mM DTT) containing an antiprotease mixture (0.5 mM DFP, 0.5 mM 4-(2 aminoethyl)benzenesulfonyl fluoride, 1 mM PMSF, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, final concentrations). After a 10-min incubation on ice, an equal volume of lysis buffer containing the antiprotease mixture as well as 0.2% NP-40 was added (to yield a final concentration of 0.1% NP-40). Samples were immediately vortex mixed for 15 s before centrifugation at 1,200 x g (5 min at 4°C). The resulting nuclear pellets were washed once with lysis buffer containing the antiprotease mixture before being resuspended in ice-cold nuclear extraction buffer (20 mM HEPES [pH 7.90], 400 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, and 10% [vol/vol] glycerol) containing the antiprotease mixture. After a 20-min incubation on ice (with frequent mixing), samples were spun (15,000 x g for 15 min at 4°C), and supernatants (the nuclear extracts) were snap-frozen in liquid nitrogen and stored at –80°C. Extracts were routinely processed for protein content determination. Nuclear extracts (amounts used are specified in the FIGURE LEGENDS) were analyzed in EMSA. The sequences of the sense strands of the oligonucleotides used for EMSA were as follows: 5'-AGT TGA GGG GAC TTT CCC AGG C-3' (NF-{kappa}B), 5'-CGC TTG ATG AGT CAG CCG GAA-3' (AP-1) from Promega [Madison, WI]) and 5'-AGT TGA GGC GAC TTT CCC AGG C-3' (NF-{kappa}B mutant) 5'-CGC TTG ATG ACT TGG CCG GAA-3' (AP-1 mutant) from Santa Cruz Biotechnology. For supershift experiments, binding reactions were conducted in the presence of specific antisera to individual c-Jun, JunB, JunD, c-Fos, FosB, CREB1, CREB2, p65 or p50 proteins (30 min at 4°C) before the addition of {gamma}-32P–labeled probes. Samples were electrophoresed on 6% acrylamide gels at 4°C in 0.5x TBE; dried gels were then exposed to Hyperfilm MP (Amersham Pharmacia Biotech) with intensifying screens at –80°C.

Luciferase Assays
293LT1 cells were plated in 12-well tissue culture plates 24 h before transfection and transiently transfected using 1.5 µl of Fugene 6 transfection reagent (Roche Diagnostics Canada) according to the manufacturer's instructions, using 0.5 µg of plasmid DNA per well. The day after transfection, cells were serum-starved overnight before stimulation with LTD4 (10 nM) or EtOH for 6 h. Cell lysates were assayed for luciferase activity using the Dual-Glo luciferase system (Promega).

ELISA
293LT1 cells were cultured in 12-well culture plates and serum-starved overnight before stimulation with LTD4 for indicated times and concentrations. Culture supernatants were carefully collected, snap-frozen in liquid nitrogen, and stored at –80°C. IL-8 concentrations were determined using the Opt-EIA Human IL-8 ELISA kit (BD Pharmingen, San Diego, CA).

Statistical Analyses
Where mentioned, statistical significance was assessed using the Student's t test for paired data (two-tailed) using PRISM4 software (GraphPad Software, San Diego, CA). Differences were considered significant at P <= 0.05 for n >= 3.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
LTD4 Induces IL-8 Gene Expression in Monocytic THP-1 Cells and Human Dendritic Cells
Mononuclear phagocytes readily express the CysLT1 receptor, and we have shown the latter to be upregulated by IL-4 and IL-13 (32). Human monocytic THP-1 cells were cultured in the presence of IL-4 (10 ng/ml) for 24 h before stimulation with LTD4 or EtOH (as a vehicle control) at graded concentrations for 3 h. As shown in Figure 1A, LTD4 induced a concentration-dependent expression of IL-8 mRNA in THP-1 cells, with a maximal effect at 10 nM of LTD4. Moreover, IL-8 expression in response to LTD4 was totally prevented by pretreatment of the cells with the selective CysLT1 antagonist MK476 (Figure 1B). We recently demonstrated the expression of the CysLT1 receptor on immature dendritic cells (31). Based on this, we studied whether these cells could enhance IL-8 expression in response to LTD4. As illustrated in Figure 1C, LTD4 induced a concentration-dependent secretion of IL-8 protein. Involvement of the CysLT1 receptor was confirmed by significant abrogation of IL-8 production in the presence of MK476 (Figure 1D). Interestingly, LTB4 induced a similar induction of IL-8 production in dendritic cells.


Figure 1
View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. Induction of IL-8 gene expression by LTD4 in THP-1 cells and human dendritic cells. THP-1 cells were cultured for 24 h in the presence of IL-4 (10 ng/ml) before addition of LTD4. (A) Northern blot analysis of IL-8 mRNA expression in THP-1 cells cultured in the presence of graded concentrations of LTD4 or vehicle (EtOH) for 3 h. The 18S RNA expression was used as an internal control. (B) Northern blot analysis of IL-8 mRNA expression in THP-1 cells preincubated in the absence or presence of the CysLT1 antagonist MK476 (1 µM) and then cultured for 3 h in the presence of 10 nM LTD4 or vehicle (EtOH). Results are representative of two separate experiments. (C) Immature monocyte-derived dendritic cells were stimulated with LTD4 or EtOH for 24 h at indicated concentrations and supernatants were collected to measure IL-8 protein by ELISA. Data are representative of two independent experiments. (D) Dendritic cells were also pretreated or not with montelukast (MK476) for 30 min and were then stimulated with LTD4 or LTB4 at indicated concentrations or with vehicle (EtOH) for 24 h. Supernatants were collected to measure IL-8 protein by ELISA. Data are expressed as fold increase relative to EtOH control (**P < 0.01 using Student's t test; n = 2).

 
These results prompted us to generate easily transfectable cells that expressed only the CysLT1 receptor to study the signaling pathways that lead to induction of IL-8 expression by LTD4. We thus produced the stably transfected 293LT1 cells, as described in MATERIALS AND METHODS.

LTD4 Stimulates IL-8 Synthesis and Release in 293LT1 Cells
Concentration-response and time-course experiments were conducted to investigate the effect of LTD4 on IL-8 expression in 293LT1 cells. LTD4 strongly upregulated IL-8 mRNA at concentrations of 10 and 100 nM (Figure 2A). IL-8 protein production was dependent on the concentration of LTD4, as illustrated in Figure 2C, with maximum induction at 10 nM of LTD4 for 8 h. Kinetics of IL-8 mRNA expression in response to LTD4 are shown in Figure 2B. Results indicate an increase in IL-8 mRNA within 2 h, with a maximum obtained after 4 h of stimulation. At 24 h, IL-8 mRNA began to decrease, indicating that the effect of LTD4 was rapid but transient. Furthermore, cells were cultured in the presence of LTD4 or EtOH for varying lengths of time; culture supernatants were collected, and their IL-8 content was analyzed by ELISA. Figure 2D illustrates the time-dependent IL-8 protein accumulation in response to LTD4, starting at 2 h and maintained through 24 h of LTD4 stimulation. As expected, selective CysLT1 antagonists (MK476 and Zafirlukast) totally abolished the IL-8 upregulation induced by LTD4 (data not shown).


Figure 2
View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. LTD4 induces a concentration- and time-dependent IL-8 synthesis in 293LT1 cells. 293LT1 cells were stimulated with LTD4 or EtOH, (A) for 4 h at indicated concentrations or (B) with 10 nM LTD4 or EtOH for indicated time periods. IL-8 mRNA expression is shown by Northern blot analysis using a specific human IL-8 probe. 293LT1 cells were also separately treated with LTD4 or EtOH, (C) for 8 h at indicated concentrations or (D) with LTD4 (10 nM) or EtOH for indicated time periods, and supernatants were collected to measure IL-8 protein by ELISA. Data are representative of three independent experiments and are expressed as the means ± SEM (n = 3).

 
LTD4 Induces IL-8 Promoter Activity in a Concentration-Dependent Manner
To determine whether LTD4 could induce IL-8 transcription, we measured the capacity of LTD4 to activate the transcription of a luciferase reporter gene driven by the IL-8 promoter (–162/+44). Kinetics of IL-8 promoter activity are shown in Figure 3A, indicating a strong induction (up to 100-fold) of IL-8 transactivation using 10 and 100 nM of LTD4.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Implication of NF-{kappa}B and AP-1 binding sites in IL-8 promoter activity stimulated by LTD4. (A) 293LT1 cells were transiently transfected with 0,5 µg/well pOLUC (promoterless vector) or pIL-8–WT promoter constructs. Cells were incubated for 6 h with EtOH or LTD4 at indicated concentrations before measurement of luciferase activity. (B) 293LT1 cells were transiently transfected with 0.5 µg/well pOLUC (promoterless vector), IL-8–WT, IL-8–{Delta}NF-{kappa}B, IL-8–{Delta}AP-1 or IL-8–{Delta}NF–IL-6 promoter constructs. Cells were incubated for 6 h with EtOH or LTD4 at 10 nM before measurement of luciferase activity. (C) 293LT1 cells were transfected with the pTA–AP-1–Luc (open bars) or pTA–NF-{kappa}B–Luc (filled bars) promoter constructs containing multimers of AP-1 or NF-{kappa}B sites, respectively. Cells were then stimulated with LTD4 for 6 h before luciferase activity measurement. Data are expressed as fold increase relative to EtOH control (*P < 0.05, **P < 0.01, or ***P < 0.001 relative to EtOH control cells; n = 3).

 
Implication of the NF-{kappa}B and AP-1 Sites in IL-8 Promoter Activity Stimulated by LTD4
The IL-8 promoter is well characterized and contains several binding sites for transcription factors, including the AP-1 (–126, –120), NF–IL-6 (–94, –81) and NF- {kappa}B (–80, –70) sites (33). To determine the implication of each site in the context of LTD4 stimulation, we used IL-8 promoter constructs containing site-specific mutations (30). As shown in Figure 3B, the NF-{kappa}B site mutation reduced the IL-8 promoter activity induced by LTD4 by 64%. Similarly, the AP-1 site mutation inhibited LTD4-induced IL-8 promoter activity by 78%. In contrast, mutation of the NF–IL-6 site had no effect. To determine whether these sites are independently inducible by LTD4, we measured the capacity of LTD4 to activate the transcription of a luciferase gene reporter containing tandem copies of consensus sequence of NF-{kappa}B or AP-1 elements. As presented in Figure 3C, LTD4 treatment resulted in a twofold increase in NF-{kappa}B and a threefold increase in AP-1–driven luciferase reporter activity, indicating independent activation of these elements by CysLTs.

Role of NF-{kappa}B Pathway in the Induction of IL-8 Promoter Activity by LTD4
Based on the previous results, we investigated which elements of the NF-{kappa}B pathway could be activated by LTD4. As shown in Figure 4A, LTD4 induced phosphorylation of I{kappa}B{alpha} as early as 5 min after stimulation, with persistence until 60 min, and a maximal induction after 15 min. As expected, total I{kappa}B{alpha} protein was reduced after LTD4 stimulation because of the known ubiquitination and proteasomal degradation of I{kappa}B{alpha} after its phosphorylation. LTD4 also stimulated a time-dependent phosphorylation on serine 536 of the p65 NF-{kappa}B subunit, with a strong activation 60 min after stimulation (Figure 4A). To determine whether I{kappa}B{alpha} phosphorylation was required for LTD4-induced IL-8 gene transcription, 293LT1 cells were transfected with a dominant negative form of I{kappa}B{alpha}, along with the IL-8 promoter–luciferase construct, and then cultured in the presence of LTD4 for 6 h. As shown in Figure 4B, overexpression of dominant negative I{kappa}B{alpha} significantly reduced IL-8 promoter activity by 54% after LTD4 stimulation.


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		Figure 4. Role of NF-{kappa}B pathway in the induction of IL-8 promoter activity by LTD4. (A) 293LT1 cells were stimulated with LTD4 or EtOH for indicated time periods. Total proteins were separated by electrophoresis and Western blotted using indicated Abs. At the bottom of the panel, total p65 protein expression served as internal control. (B) Transient transfection of 293LT1 cells with 0.5 µg/well pIL-8–WT and pcDNA or dominant negative I{kappa}B{alpha} construct. Cells were incubated for 6 h with EtOH or LTD4 at 10 nM before measurement of luciferase activity. Data are expressed as fold increase relative to EtOH control (***P < 0.001 using Student t test; n = 3).

 
Effect of LTD4 on NF-{kappa}B DNA Binding Activity
Because we saw involvement of the NF-{kappa}B pathway, we studied whether NF-{kappa}B DNA binding was induced by LTD4. As shown in Figure 5A, exposure of 293LT1 cells to 10 nM of LTD4 for graded periods of time resulted in time-dependent induction of NF-{kappa}B DNA binding activity, which could be observed within 15 min of stimulation. To study the nature of the complex induced by LTD4, supershift experiments were performed using specific Abs. Results in Figure 5B indicate that the NF-{kappa}B binding complex induced by LTD4 was composed of p50 and p65.


Figure 5
View larger version (62K):
[in this window]
[in a new window]
  		Figure 5. Effect of LTD4 on the activation of nuclear NF-{kappa}B DNA binding activities. (A) 293LT1 cells were cultured for the indicated times in the presence of LTD4 (10 nM) or EtOH before nuclear extraction and EMSA analysis. (B) 293LT1 cells were stimulated for 60 min before nuclear extract preparation. The nuclear extracts were analyzed in EMSA using a radiolabeled consensus NF-{kappa}B oligonucleotide probe. The specificity of complex formation was tested by the inclusion of unlabeled competitors (cold probe, lane 6; or cold mutated probe, lane 7) by including specific anti-p50 (lane 3), anti-p65 (lane 4), or an isotype-matched control Abs (lane 5).

 
LTD4 Regulates c-fos and c-jun mRNA Expression
We also investigated whether LTD4 stimulation leads to the transcriptional regulation of AP-1 subunits, namely, the c-Fos and c-Jun protooncogenes. As shown in Figure 6A, LTD4 induced a rapid and transient expression of c-fos mRNA. After LTD4 stimulation, c-fos mRNA was detectable within 15 min, reaching maximal expression at 30 and 45 min, and returning to basal levels by 60 min. Figure 6B illustrates a similar pattern of expression for c-jun mRNA after LTD4 stimulation. Furthermore, kinetics of phosphorylation indicated a strong activation of the c-Jun protein after LTD4 stimulation (data not shown).


Figure 6
View larger version (44K):
[in this window]
[in a new window]
  		Figure 6. LTD4 upregulates c-fos and c-jun mRNA expression. 293LT1 cells were stimulated with LTD4 10 nM or EtOH for indicated time periods. Total cellular RNA was then extracted, separated by electrophoresis, and Northern blotted using c-fos (A) and c-jun (B) probes. Results are representative of two separate experiments.

 
LTD4 Increases AP-1 DNA Binding Activity
We next examined whether treatment of 293LT1 cells with LTD4 modulated the AP-1 DNA binding activity. Figure 7A shows the kinetics of AP-1 activation by LTD4, indicating a detectable enhancement within 30 min and strong induction at 60 min. Analysis of the binding complex was done after 60 min of stimulation using selected Abs. As shown in Figure 7B, the LTD4-inducible complex contained c-Fos and c-Jun, because it could be supershifted using Abs that recognize specifically these proteins, but not by Abs to JunB, JunD, FosB, CREB1, CREB2, or control IgG.


Figure 7
View larger version (64K):
[in this window]
[in a new window]
  		Figure 7. Analysis of AP-1 binding after LTD4 stimulation. (A) 293LT1 cell were cultured in the presence of LTD4 (10 nM) or EtOH for indicated times before nuclear extract preparation. The nuclear extracts were analyzed in EMSA using a radiolabeled consensus AP-1 oligonucleotide probe. (B) Cells were stimulated with 10 nM LTD4 or EtOH for 60 min before EMSA analysis. The specificity of complex formation was tested by the inclusion of unlabeled competitors (cold probe, lane 11; or cold mutated probe, lane 12) by including specific anti-c-Jun (lane 3), anti-JunD (lane 4), anti-JunB (lane 5), anti-c-Fos (lane 6), anti-FosB (lane 7), anti-CREB1 (lane 8), anti-CREB2 (lane 9), or an isotype-matched control Abs (lane 10).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
LTD4 is known to play a major role in the pathogenesis of asthma. Inflammation is an important characteristic of the late-phase reaction of asthma, and is heralded by cellular infiltration in the lung. At this point, little is known about the mechanism of inflammation orchestrated by CysLTs in asthma. Here we report a direct action of LTD4 on the transcriptional modulation of IL-8 expression. IL-8 is a potent chemoattractant for neutrophils and plays a major role in promoting inflammation, as found in severe asthma (15, 16, 34) and COPD (19, 20). Elevated expression levels of IL-8 have been reported in BALF and sputum of patients with asthma (15, 16). Our results demonstrate that LTD4, acting through the CysLT1 receptor, can induce the expression of IL-8. The effect is time- and concentration-dependent. We also studied the phenomenon in the human monocytic cell line, THP-1, in which LTD4 induced a concentration-dependent expression of IL-8 mRNA. As in the 293LT1 model, nanomolar concentrations of LTD4 induced the highest levels of IL-8 gene expression. Interestingly, immature dendritic cells also produced increased amounts of IL-8 in response to LTD4. Furthermore, the specific CysLT1 antagonist, MK476, completely prevented the modulation of IL-8 mRNA in THP-1 and of IL-8 protein in dendritic cells, indicating that the effect induced by LTD4 was mediated through the CysLT1 receptor. We and others have previously reported the expression of CysLT1 receptors on leukocytes, including alveolar and monocyte-derived macrophages, as well as dendritic cells and B lymphocytes (6, 8, 31, 32, 35). Moreover, CysLT1 expression levels were upregulated by IL-13 and IL-4 in monocytes and macrophages, and the increased CysLT1 receptor expression resulted in enhanced Ca2+ transients and chemotactic responses to LTD4 (32).

The functional and clinical relevance of our findings may lie in the interplay between CysLTs, chemokines, neutrophils, and eosinophils, as reported in several pathologies. The CysLT1 antagonists montelukast and pranlukast were recently shown to prevent neutrophil infiltration in models of ischemia–reperfusion of the kidney (36), sepsis-induced hepatic and ileal injury (37), and brain ischemia (38). Moreover, montelukast was found to decrease serum and sputum levels of IL-8 and bronchial levels of eosinophil and neutrophil infiltration in children with cystic fibrosis (39). Interestingly, Kikuchi and colleagues (40) recently demonstrated that IL-8–exposed neutrophils induced eosinophil trans–basement membrane migration.

The signaling mechanisms elicited by CysLT1 are still poorly defined and have not been reported in the context of modulation of IL-8 expression. Our studies initially demonstrated that LTD4-induced IL-8 release is transcriptionally regulated, using a reporter gene assay. Based on a well characterized IL-8 promoter sequence, mutation of different binding sites for transcription factors was used to initiate our signaling experiments. The NF-{kappa}B site is known as an important factor in the induction of IL-8 gene expression by different proinflammatory factor, such as TNF-{alpha} and IL-1beta (41). Several laboratories have demonstrated that G-protein–coupled receptors are able to activate the NF-{kappa}B pathway. For example, IL-6 expression induced by LTB4 (42) and monocyte chemotactic protein–1 released by platelet-activating factor stimulation (43) are mediated by activation of NF-{kappa}B. In this respect, our results showed that NF-{kappa}B site mutation reduced the IL-8 promoter activity induced by LTD4 by 64%. Moreover, the role of AP-1 in the induction of IL-8 gene expression by LTD4 was demonstrated by a 78% reduction of the promoter activity when the AP-1 transcription site was mutated. Conversely, the NF–IL-6 site, known for its cooperation with NF-{kappa}B elements (44, 45), does not appear to be involved in LTD4-treated 293LT1 cells, because mutation in the NF–IL-6 site did not affect IL-8 promoter activity. In summary, our data showed that NF-{kappa}B and AP-1 were necessary for induction of IL-8 transactivation in response to LTD4.

Cell populations that express CysLT1 usually also express CysLT2. Whereas there are selective antagonists for CysLT1, already in clinical use for asthma treatment, there is no selective antagonist for CysLT2, and compound BAY-u9773 is a weak dual antagonist, but also a partial CysLT2 agonist. Transfected cell lines stably expressing only one of the receptors provide a useful tool to dissect selective receptor-mediated signaling mechanisms. As expected, selective antagonists of CysLT1 completely prevented the IL-8 transactivation induced by LTD4 in 293LT1 cells. Interestingly, recent reports have suggested that pranlukast and MK-571, two selective CysLT1 antagonists, could suppress NF-{kappa}B activation independently of CysLT1 receptor antagonism (46–48). These reports underscore the interest of using our model, the stably transfected 293LT1 cells, as a tool to specifically study CysLT1 signaling. Moreover, additional genes that are also activated by the same transcription factors after CysLT1 receptor signaling can be identified. In this regard, preliminary results from our laboratory indicate that the chemokine CCL2 (monocyte chemotactic protein–1), another gene that is regulated in part by NF-{kappa}B (49), can also be upregulated by LTD4 in 293LT1 cells and THP-1 cells (unpublished data).

In resting cells, the NF-{kappa}B complex is found in the cytoplasm in an inactive state, bound to the inhibitory protein I{kappa}B through association with p65 (RelA) (50). In response to various stimuli, the I{kappa}B{alpha} subunit is first phosphorylated at serines 32 and 36 by I{kappa}B kinase, rapidly polyubiquitinated and degraded through the 26S proteasome, allowing translocation of free NF-{kappa}B to the nucleus and subsequent gene activation (51, 52). Our results indicate that the consequence of LTD4 stimulation was the phosphorylation of IkB{alpha} followed by the proteolysis of IkB{alpha} protein and simultaneous activation of the p65 NF-{kappa}B, as demonstrated by phosphorylation on serine 536. This finding is consistent with another report demonstrating the activation of NF-{kappa}B p65 in isolated lung monocytes in a murine model of allergic asthma (53). In our model, overexpression of a dominant negative form of I{kappa}B{alpha} also suggested the implication of I{kappa}B{alpha} in the transactivation of IL-8 promoter induced by LTD4. NF-{kappa}B can be found as a p50 homodimer or, in most cell types, as a heterodimer composed of p65 and p50 (25). Our data show that nuclear factor binding to the NF-{kappa}B element was strongly increased in LTD4-treated cells, with formation of a complex that included p50 and p65, as indicated by a supershift in EMSA. Interestingly, NF-{kappa}B is involved in the expression of a variety of genes controlling immune and inflammatory responses, including early response genes encoding for cytokines, tissue factor, and adhesion molecules. Some reports established a correlation between NF-{kappa}B pathway activation and asthma exacerbation (54, 55).

We also examined whether AP-1 played a role in the CysLT1 signaling. The transcription factor AP-1 is a target of interest because of its cognate sequence within the IL-8 promoter known to participate in the transcriptional induction. AP-1 is a heterodimer consisting of members of the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD) families of transcription factors (56). The AP-1 complex is a homodimer of Jun proteins or a heterodimer of Jun and Fos molecules. Regulation of AP-1 activity is critically dependent on the relative proportion of its subunits present in the cells (57). Here we showed for the first time a rapid and transient upregulation of c-fos and c-jun expression by LTD4 at the mRNA level. The kinetics of expression of these two transcription factors were similar. AP-1 elements are also regulated at post-translational levels, characterized by phosphorylation of its subunits. We observed a strong phosphorylation of c-Jun 60 min after LTD4 stimulation. The protooncogenes c-fos and c-jun are recognized as early-immediate genes, playing a role in cell cycle. c-Jun:c-Jun homodimer complexes or c-Fos:c-Jun heterodimers have been found to regulate the expression of many genes including c-Jun (58) and COX-2 (59). In our studies, supershift assays showed the formation of a c-Fos:c-Jun complex after LTD4 stimulation. This also constitutes the first demonstration that LTD4 can induce AP-1 activation and is consistent with the involvement of AP-1 in IL-8 promoter activation.

In summary, we demonstrated that NF-{kappa}B and AP-1 play a key role in the signaling driven by the CysLT1 receptor that leads to the induction of IL-8 expression. The current study suggests that LTD4 not only acts as a direct chemotactic factor, but may also promote the production of IL-8, a chemokine that activates a number of inflammatory cells types. In a broader context, it may be important to consider the potential for LTD4 to induce the expression of a variety of proinflammatory genes through its ability to activate AP-1 and NF-{kappa}B.


   Acknowledgments
 
The authors thank Dr. Allan R. Brasier (University of Texas) for the IL-8 promoter constructs, Dr. Christian Jobin (University of North Carolina at Chapel Hill) for the dominant negative form of I{kappa}B{alpha}, and Dr. Claude Asselin (University of Sherbrooke) for the human c-Jun probe.


   Footnotes
 
This work was supported by grants (P.P.McD., J.S., and M.R.P.) and studentships (C.T., A.C., and Y.B.) from the Canadian Institutes of Health Research. P.P.McD. is a scholar of the Fonds de la recherche en santé du Québec and M.R.P. is the awardee of a Canada Research Chair in Inflammation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0407OC on July 29, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 3, 2005

Accepted in final form June 26, 2006


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Devillier P, Baccard N, Advenier C. Leukotrienes, leukotriene receptor antagonists and leukotriene synthesis inhibitors in asthma: an update. Part II: clinical studies with leukotriene receptor antagonists and leukotriene synthesis inhibitors in asthma. Pharmacol Res 1999;40:15–29.[CrossRef][Medline]
  2. Wenzel SE, Larsen GL, Johnston K, Voelkel NF, Westcott JY. Elevated levels of leukotriene C4 in bronchoalveolar lavage fluid from atopic asthmatics after endobronchial allergen challenge. Am Rev Respir Dis 1990;142:112–119.[Medline]
  3. Lam S, Chan H, LeRiche JC, Chan-Yeung M, Salari H. Release of leukotrienes in patients with bronchial asthma. J Allergy Clin Immunol 1988;81:711–717.[CrossRef][Medline]
  4. Macfarlane AJ, Dworski R, Sheller JR, Pavord ID, Kay AB, Barnes NC. Sputum cysteinyl leukotrienes increase 24 hours after allergen inhalation in atopic asthmatics. Am J Respir Crit Care Med 2000;161: 1553–1558.[Abstract/Free Full Text]
  5. Pavord ID, Ward R, Woltmann G, Wardlaw AJ, Sheller JR, Dworski R. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med 1999;160:1905–1909.[Abstract/Free Full Text]
  6. Lynch KR, O'Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueroa DJ, Zeng Z, et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 1999;399:789–793.[CrossRef][Medline]
  7. Heise CE, O'Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, Cheng R, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 2000;275: 30531–30536.[Abstract/Free Full Text]
  8. Figueroa DJ, Breyer RM, Defoe SK, Kargman S, Daugherty BL, Waldburger K, Liu Q, Clements M, Zeng Z, O'Neill GP, et al. Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 2001;163: 226–233.[Abstract/Free Full Text]
  9. Labat C, Ortiz JL, Norel X, Gorenne I, Verley J, Abram TS, Cuthbert NJ, Tudhope SR, Norman P, Gardiner P, et al. A second cysteinyl leukotriene receptor in human lung. J Pharmacol Exp Ther 1992;263: 800–805.[Abstract/Free Full Text]
  10. Lewis RA, Austen KF, Soberman RJ. Leukotrienes and other products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiology in human diseases. N Engl J Med 1990;323:645–655.[Medline]
  11. Drazen JM. Leukotrienes as mediators of airway obstruction. Am J Respir Crit Care Med 1998;158:S193–S200.[Abstract/Free Full Text]
  12. Spada CS, Krauss AH, Nieves AL, Woodward DF. Effects of leukotrienes B4 (LTB4) and D4 (LTD4) on motility of isolated normodense human eosinophils and neutrophils. Adv Exp Med Biol 1997;400B:699–706.
  13. Pizzichini E, Leff JA, Reiss TF, Hendeles L, Boulet LP, Wei LX, Efthimiadis AE, Zhang J, Hargreave FE. Montelukast reduces airway eosinophilic inflammation in asthma: a randomized, controlled trial. Eur Respir J 1999;14:12–18.[Abstract]
  14. Minoguchi K, Kohno Y, Minoguchi H, Kihara N, Sano Y, Yasuhara H, Adachi M. Reduction of eosinophilic inflammation in the airways of patients with asthma using montelukast. Chest 2002;121:732–738.[Medline]
  15. Ordonez CL, Shaughnessy TE, Matthay MA, Fahy JV. Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma: clinical and biologic significance. Am J Respir Crit Care Med 2000;161:1185–1190.[Abstract/Free Full Text]
  16. Gibson PG, Simpson JL, Saltos N. Heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin-8. Chest 2001;119:1329–1336.[CrossRef][Medline]
  17. Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K. Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol 1994;56:559–564.[Abstract]
  18. Lemiere C, Pelissier S, Tremblay C, Chaboillez S, Thivierge M, Stankova J, Rola-Pleszczynski M. Leukotrienes and isocyanate-induced asthma: a pilot study. Clin Exp Allergy 2004;34:1684–1689.[CrossRef][Medline]
  19. Beeh KM, Kornmann O, Buhl R, Culpitt SV, Giembycz MA, Barnes PJ. Neutrophil chemotactic activity of sputum from patients with COPD: role of interleukin 8 and leukotriene B4. Chest 2003;123:1240–1247.[CrossRef][Medline]
  20. Pesci A, Balbi B, Majori M, Cacciani G, Bertacco S, Alciato P, Donner CF. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J 1998;12:380–386.[Abstract]
  21. Hay DW, Sarau HM. Interleukin-8 receptor antagonists in pulmonary diseases. Curr Opin Pharmacol 2001;1:242–247.[CrossRef][Medline]
  22. Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin-8 gene expression. J Leukoc Biol 2002;72:847–855.[Abstract/Free Full Text]
  23. Zingarelli B, Sheehan M, Wong HR. Nuclear factor-{kappa}B as a therapeutic target in critical care medicine. Crit Care Med 2003;31:S105–S111.[CrossRef][Medline]
  24. Hayden MS, Ghosh S. Signaling to NF-{kappa}B. Genes Dev 2004;18:2195–2224.[Abstract/Free Full Text]
  25. Senftleben U, Karin M. The IKK/NF-{kappa}B pathway. Crit Care Med 2002;30:S18–S26.[CrossRef][Medline]
  26. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1991;1072: 129–157.[Medline]
  27. Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 2004;117:5965–5973.[Abstract/Free Full Text]
  28. Levy BD, Bonnans C, Silverman ES, Palmer LJ, Marigowda G, Israel E. Diminished lipoxin biosynthesis in severe asthma. Am J Respir Crit Care Med 2005;172:824–830.[Abstract/Free Full Text]
  29. Green SA, Malice MP, Tanaka W, Tozzi CA, Reiss TF. Increase in urinary leukotriene LTE4 levels in acute asthma: correlation with airflow limitation. Thorax 2004;59:100–104.[Abstract/Free Full Text]
  30. Vlahopoulos S, Boldogh I, Casola A, Brasier AR. Nuclear factor-{kappa}B–dependent induction of interleukin-8 gene expression by tumor necrosis factor {alpha}: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation. Blood 1999;94:1878–1889.[Abstract/Free Full Text]
  31. Thivierge M, Stankova J, Rola-Pleszczynski M. Toll-like receptor agonists differentially regulate cysteinyl-leukotriene receptor 1 expression and function in human dendritic cells. J Allergy Clin Immunol 2006;117: 1155–1162.[CrossRef][Medline]
  32. Thivierge M, Stankova J, Rola-Pleszczynski M. IL-13 and IL-4 up-regulate cysteinyl leukotriene 1 receptor expression in human monocytes and macrophages. J Immunol 2001;167:2855–2860.[Abstract/Free Full Text]
  33. Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J Immunol 1989;143:1366–1371.[Abstract]
  34. Wenzel SE, Szefler SJ, Leung DY, Sloan SI, Rex MD, Martin RJ. Bronchoscopic evaluation of severe asthma: persistent inflammation associated with high dose glucocorticoids. Am J Respir Crit Care Med 1997; 156:737–743.[Abstract/Free Full Text]
  35. Lamoureux J, Stankova J, Rola-Pleszczynski M. Leukotriene D4 enhances immunoglobulin production in CD40-activated human B lymphocytes. J Allergy Clin Immunol 2006;117:924–930.[CrossRef][Medline]
  36. Sener G, Sehirli O, Velioglu-Ogunc A, Cetinel S, Gedik N, Caner M, Sakarcan A, Yegen BC. Montelukast protects against renal ischemia/reperfusion injury in rats. Pharmacol Res 2006;54:65–71.[CrossRef][Medline]
  37. Sener G, Sehirli O, Cetinel S, Ercan F, Yuksel M, Gedik N, Yegen BC. Amelioration of sepsis-induced hepatic and ileal injury in rats by the leukotriene receptor blocker montelukast. Prostaglandins Leukot Essent Fatty Acids 2005;73:453–462.[CrossRef][Medline]
  38. Chu LS, Wei EQ, Yu GL, Fang SH, Zhou Y, Wang ML, Zhang WP. Pranlukast reduces neutrophil but not macrophage/microglial accumulation in brain after focal cerebral ischemia in mice. Acta Pharmacol Sin 2006;27:282–288.[CrossRef][Medline]
  39. Schmitt-Grohe S, Zielen S. Leukotriene receptor antagonists in children with cystic fibrosis lung disease: anti-inflammatory and clinical effects. Paediatr Drugs 2005;7:353–363.[CrossRef][Medline]
  40. Kikuchi I, Kikuchi S, Kobayashi T, Hagiwara K, Sakamoto Y, Kanazawa M, Nagata M. Eosinophil trans-basement membrane migration induced by interleukin-8 and neutrophils. Am J Respir Cell Mol Biol 2006;34:760–765.[Abstract/Free Full Text]
  41. Mukaida N, Mahe Y, Matsushima K. Cooperative interaction of nuclear factor–{kappa} B– and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem 1990;265:21128–21133.[Abstract/Free Full Text]
  42. Brach MA, de Vos S, Arnold C, Gruss HJ, Mertelsmann R, Herrmann F. Leukotriene B4 transcriptionally activates interleukin-6 expression involving NK-{chi} B and NF–IL6. Eur J Immunol 1992;22:2705–2711.[Medline]
  43. Weyrich AS, McIntyre TM, McEver RP, Prescott SM, Zimmerman GA. Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-{alpha} secretion: signal integration and NF-{kappa} B translocation. J Clin Invest 1995;95:2297–2303.[Medline]
  44. Stein B, Baldwin AS Jr. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-{kappa} B. Mol Cell Biol 1993;13:7191–7198.[Abstract/Free Full Text]
  45. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF–IL6 and NF-{kappa} B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 1993;90: 10193–10197.[Abstract/Free Full Text]
  46. Ichiyama T, Hasegawa S, Umeda M, Terai K, Matsubara T, Furukawa S. Pranlukast inhibits NF-{kappa} B activation in human monocytes/macrophages and T cells. Clin Exp Allergy 2003;33:802–807.[CrossRef][Medline]
  47. Tomari S, Matsuse H, Machida I, Kondo Y, Kawano T, Obase Y, Fukushima C, Shimoda T, Kohno S. Pranlukast, a cysteinyl leukotriene receptor 1 antagonist, attenuates allergen-specific tumour necrosis factor {alpha} production and nuclear factor {kappa} B nuclear translocation in peripheral blood monocytes from atopic asthmatics. Clin Exp Allergy 2003; 33:795–801.[CrossRef][Medline]
  48. Ishinaga H, Takeuchi K, Kishioka C, Suzuki S, Basbaum C, Majima Y. Pranlukast inhibits NF-{kappa}B activation and MUC2 gene expression in cultured human epithelial cells. Pharmacology 2005;73:89–96.[CrossRef][Medline]
  49. Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T. NF-{kappa} B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 1994;153:2052–2063.[Abstract]
  50. Baeuerle PA, Baltimore D. A 65-{kappa}D subunit of active NF-{kappa}B is required for inhibition of NF-{kappa}B by I {kappa}B. Genes Dev 1989;3:1689–1698.[Abstract/Free Full Text]
  51. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-{kappa} B1 precursor protein and the activation of NF-{kappa} B. Cell 1994;78:773–785.[CrossRef][Medline]
  52. Traenckner EB, Pahl HL, Henkel T, Schmidt KN, Wilk S, Baeuerle PA. Phosphorylation of human I {kappa} B-{alpha} on serines 32 and 36 controls I {kappa} B-{alpha} proteolysis and NF-{kappa} B activation in response to diverse stimuli. EMBO J 1995;14:2876–2883.[Medline]
  53. Kawano T, Matsuse H, Kondo Y, Machida I, Saeki S, Tomari S, Mitsuta K, Obase Y, Fukushima C, Shimoda T, et al. Cysteinyl leukotrienes induce nuclear factor {kappa} B activation and RANTES production in a murine model of asthma. J Allergy Clin Immunol 2003;112:369–374.[CrossRef][Medline]
  54. Gagliardo R, Chanez P, Mathieu M, Bruno A, Costanzo G, Gougat C, Vachier I, Bousquet J, Bonsignore G, Vignola AM. Persistent activation of nuclear factor–{kappa}B signaling pathway in severe uncontrolled asthma. Am J Respir Crit Care Med 2003;168:1190–1198.[Abstract/Free Full Text]
  55. Hart LA, Krishnan VL, Adcock IM, Barnes PJ, Chung KF. Activation and localization of transcription factor, nuclear factor–{kappa}B, in asthma. Am J Respir Crit Care Med 1998;158:1585–1592.[Abstract/Free Full Text]
  56. Kaminska B, Pyrzynska B, Ciechomska I, Wisniewska M. Modulation of the composition of AP-1 complex and its impact on transcriptional activity. Acta Neurobiol Exp (Wars) 2000;60:395–402.[Medline]
  57. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240–246.[CrossRef][Medline]
  58. Angel P, Hattori K, Smeal T, Karin M. The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell 1988;55:875–885.[CrossRef][Medline]
  59. Chen LC, Chen BK, Chang JM, Chang WC. Essential role of c-Jun induction and coactivator p300 in epidermal growth factor-induced gene expression of cyclooxygenase-2 in human epidermoid carcinoma A431 cells. Biochim Biophys Acta 2004;1683:38–48.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C. Thompson, S. McMahon, Y. Bosse, C. M. Dubois, J. Stankova, and M. Rola-Pleszczynski
Leukotriene D4 Up-Regulates Furin Expression through CysLT1 Receptor Signaling
Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 227 - 234.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Woszczek, L.-Y. Chen, S. Nagineni, and J. H. Shelhamer
IL-10 Inhibits Cysteinyl Leukotriene-Induced Activation of Human Monocytes and Monocyte-Derived Dendritic Cells
J. Immunol., June 1, 2008; 180(11): 7597 - 7603.
[Abstract] [Full Text] [PDF]

Home page
 ChestHome page
J. Shannon, P. Ernst, Y. Yamauchi, R. Olivenstein, C. Lemiere, S. Foley, L. Cicora, M. Ludwig, Q. Hamid, and J. G. Martin
Differences in Airway Cytokine Profile in Severe Asthma Compared to Moderate Asthma
Chest, February 1, 2008; 133(2): 420 - 426.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Thompson, A. Cloutier, Y. Bosse, C. Poisson, P. Larivee, P. P. McDonald, J. Stankova, and M. Rola-Pleszczynski
Signaling by the Cysteinyl-Leukotriene Receptor 2: INVOLVEMENT IN CHEMOKINE GENE TRANSCRIPTION
J. Biol. Chem., January 25, 2008; 283(4): 1974 - 1984.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0407OCv1
35/6/697    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Thompson, C.
Right arrow