help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 14, 2003, doi:10.1165/rcmb.2003-0071OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0071OCv1
30/2/212    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 Steinke, J. W.
Right arrow Articles by Borish, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinke, J. W.
Right arrow Articles by Borish, L.
American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 212-219, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0071OC

Characterization of Interleukin-4–Stimulated Nasal Polyp Fibroblasts

John W. Steinke, Charles D. Crouse, Dewayne Bradley, Kathleen Hise, Kevin Lynch, Stilianos E. Kountakis and Larry Borish

Departments of Medicine, Pharmacology, and Otolaryngology, Asthma and Allergic Disease Center, Beirne Carter Center for Immunology Research, University of Virginia Health System, Charlottesville, Virginia

Address correspondence to: John W. Steinke, Ph.D., Asthma and Allergic Disease Center, Box 801355, University of Virginia Health System, Charlottesville, VA 22908. Email: js3ch{at}virginia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic hyperplastic eosinophilic sinusitis is an inflammatory disease that results in the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells at the site of injury. A common feature of this disease is the presence of nasal polyposis (NP). The current studies were designed to assess the contribution of interleukin (IL)-4 to fibroblast-mediated inflammation in chronic hyperplastic eosinophilic sinusitis/NP. In addition, we hypothesized that cysteinyl leukotrienes (CysLT) may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. Fibroblasts were isolated from NP tissue. All fibroblast lines expressed the IL-4 receptor. IL-4 induced changes in mRNA and protein expression of fibrotic (transforming growth factor-ß1 and -ß2) and inflammatory cytokines and chemokines (IL-6 and CCL11) by fibroblasts as measured by semiquantitative and quantitative polymerase chain reaction, RNase protection assay, and enzyme-linked immunosorbent assay. The expression of CysLT and other proinflammatory lipid receptors on fibroblasts was evaluated. CysLT1 and CysLT2 receptors were not expressed on fibroblasts; however, LPA1 receptor was constitutively expressed and LPA2 receptor expression was upregulated by IL-4. The metabolic cascade involved in CysLT synthesis was not expressed in fibroblasts and could not be induced by IL-4 treatment.

Abbreviations: chronic hyperplastic eosinophilic sinusitis, CHES • cyclooxygenase, COX • cysteinyl leukotriene, CysLT • enzyme-linked immunosorbent assay, ELISA • fetal bovine serum, FBS • 5-lipoxygenase activating protein, FLAP • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • granulocyte macrophage–colony-stimulating factor, GM-CSF • interferon, IFN • interleukin, IL • 5-lipoxygenase, 5-LO • latency-associated protein, LAP • lysophosphatidic acid, LPA • leukotriene, LT • nasal polyposis, NP • ostiomeatal complex, OMC • phosphate-buffered saline, PBS • phospholipase A2, PLA2 • ribonuclease protection assay, RPA • reverse transcriptase–polymerase chain reaction, RT-PCR • transforming growth factor, TGF • tumor necrosis factor, TNF • vascular cell adhesion molecule, VCAM


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic hyperplastic eosinophilic sinusitis (CHES), which often presents in combination with nasal polyposis (NP), is an immune inflammatory disease characterized by the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells (1, 2). The primary hallmark of this condition is considered to be the accumulation of activated eosinophils within the sinus tissue, which account for up to 28% of the inflammatory cells (3, 4). As with asthma, this disorder is associated with the activation of Th2-like lymphocytes characterized by the production of interleukin (IL)-3, IL-4, IL-5, IL-13, CCL11 (eotaxin-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other cytokines and chemokines that may contribute to this eosinophilic inflammatory disorder (5–7). CHES is characterized by the presence of cysteinyl leukotrienes (CysLTs) in polyp tissue (8), which may also contribute to eosinophil accumulation and activation (9, 10). Fibroblasts are found in the stroma of the nasal polyp and are actively involved in the accumulation of the extracellular matrix (11). The production of transforming growth factor (TGF)-{alpha}, TGF-ß, platelet-derived growth factors, heparin-binding epidermal growth factor, fibroblast growth factor, and other growth factors by activated eosinophils and these other cells may contribute to the proliferation of fibroblasts and the deposition of connective tissue observed in CHES (12).

IL-4 is a key cytokine in the development of allergic inflammation. IL-4 increases the expression of CCL11 and other inflammatory cytokines that may contribute to inflammation and remodeling in chronic asthma (13). An important activity of IL-4 in promoting cellular inflammation in the asthmatic lung is the induction of vascular cell adhesion molecule (VCAM-1) on vascular endothelium (14, 15). Through their interaction of VCAM-1, IL-4 is able to direct the migration of T lymphocytes, monocytes, basophils, and eosinophils to inflammatory loci. In addition, IL-4 inhibits eosinophil apoptosis and promotes eosinophilic inflammation by inducing eosinophil chemotaxis and activation (16). IL-4 has the ability to drive the differentiation of naive Th0 lymphocytes into Th2 lymphocytes (17, 18). These Th2 cells are able to secrete IL-4, IL-5, IL-9, and IL-13, but lose the ability to produce interferon (IFN)-{gamma} (19). IL-4 is also important to Th2-mediated immune responses through the ability to prevent apoptosis of T lymphocytes. Activation of these cells results in rapid proliferation and secretion of cytokines; however, in the absence of an appropriate signal, which can be provided by IL-4, activated T helper lymphocytes rapidly become apoptotic and are eliminated. Finally, IL-4 has been shown to upregulate expression of the CysLT receptors (20) as well as increasing the expression of leukotriene (LT) C4 synthase, a key enzyme involved in LT synthesis (21), and may thereby may contribute to the role of CysLT in this disease (8, 22).

The current studies were therefore designed to assess the contribution of IL-4 to fibroblast-mediated inflammation in CHES/NP. We evaluated IL-4–induced changes in mRNA and protein expression of fibrotic and inflammatory cytokines and chemokines by fibroblasts. In addition, we hypothesized that CysLT may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. We therefore evaluated the expression of CysLT and other proinflammatory lipid receptors on fibroblasts. These studies evaluated the effects of IL-4 on the expression of the CysLT metabolic cascade on fibroblasts. IL-13 is a cytokine closely related to IL-4 and shares many of its biological activities. Recently, IL-13 was shown to increase CysLT1 receptors on lung fibroblasts (23). We therefore compared the effects of IL-4 and IL-13 on CysLT receptor expression on NP fibroblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Polyp tissue was obtained from subjects referred to the University of Virginia Health System for sinus surgery. Subjects were asked their permission and informed consent was given to study pathologic specimens under a protocol approved by the human investigations committee at the University of Virginia Health System. No individual was studied unless he or she had been recommended for and agreed to undergo polypectomy. Study subjects were selected on the basis of a medical history consistent with chronic sinusitis, findings on rhinoscopy, and documented disease in their sinuses as shown via CT scan. Infectious sinusitis as determined by evaluating sinus tissue for neutrophils was an exclusion criterion. Similarly, additional exclusion criteria were the presence of cystic fibrosis or an immunodeficiency. Patients were not enrolled if they received oral steroids or an LT modifier within 4 wk of surgery.

Isolation of Fibroblasts from Polyp and Control Tissue
Surgical tissue was minced with scissors and placed into a 250-ml trypsinizing flask with 25 ml of tryspin-EDTA (Invitrogen, Carlsbad, CA). Samples were placed in a 37°C water bath and stirred for 30 min. An additional 25 ml of trypsin-EDTA was added to the flask and stirred again for 30 min. Fetal bovine serum (FBS; Hyclone, Logan, UT) was added to the flask to neutralize the trypsin and the tissue suspension was then centrifuged to pellet the digested tissue. Cells were resuspended in 20 ml of RPMI (Invitrogen) supplemented with 10% FBS and penicillin-streptomycin. After 1 d in culture the cells were washed to remove debris and unattached cells. Fibroblasts were grown in RPMI 1640 medium supplemented (Invitrogen) with 10% FBS (Hyclone) and antibiotics incubated in 5% CO2 at 37°C until confluent for use in assays. For assays with IL-4 and IL-13 stimulation, fibroblasts were stimulated with 10 ng/ml of cytokine (BD Bioscience, San Diego, CA) for 24 h before cells were harvested for RNA assays or 72 h for collection of supernatants.

Cytokine/Chemokine Determination
CCL11 (eotaxin-1), CXCL8 (IL-8), IL-6, and TGF-ß levels were measured in the supernatants from unstimulated or IL-4–stimulated fibroblast cultures using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (CCL11, CXCL8, IL-6, and TGF-ß; BioSource, Camarillo, CA). Fibronectin levels were measured as fibronectin bound to the cell surface in unstimulated or IL-4–stimulated fibroblast cultures using ELISA (Biomedical Technologies Inc, Stoughton, MA). The sensitivities of these assays were < 7.8 pg/ml for CCL11, < 0.39 pg/ml for CXCL8, < 0.16 pg/ml for IL-6, < 10 pg/ml for TGF-ß1, and < 25 ng/ml for fibronectin. Concentrations were calculated on the basis of standard curves using the KC4 Kineticalc for Windows software (Bio-Tek, Winooski, VT).

Reverse Transcription of mRNA
Total RNA was extracted from the fibroblast cells using a SV Total RNA Isolation kit (Promega, Madison, WI). Conversion of the mRNA to cDNA was performed using a Taqman Reverse Transcription kit (Roche, Branchburg, NJ). Briefly, 200 ng of RNA was added to each reaction along with oligo dT primers, 5.5 mM MgCl2, 2 mM dNTPs, RNasin, and reverse transcriptase. Reactions went through one cycle of 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C in a Bio-Rad iCycler thermocycler (Bio-Rad, Hercules, CA). The cDNA was amplified by polymerase chain reaction (PCR) using the appropriate primer pairs (Table 1), and the resulting products analyzed by agarose gel electrophoresis with a Kodak EDAS imaging system (Eastman Kodak, Rochester, NY). The PCR products were designed to cross exon/intron junctions, and the predicted size PCR products were found with no contamination due to genomic DNA for each primer pair. PCR reactions went through 34 cycles of 60 s each at 95°C, 55°C, and 72°C. Data were analyzed as the ratio of each cytokine to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) allowing comparison of unstimulated and stimulated fibroblasts. The primer pairs for GM-CSF and CXCL8 were purchased from Clontech (Palo Alto, CA).


View this table:
[in this window]
[in a new window]
  		TABLE 1 PCR primer pairs

 
Real-Time PCR Quantification of mRNA Levels
We used real-time PCR to quantify CCL11 and TGF-ß mRNA levels from unstimulated and IL-4–stimulated fibroblasts. The housekeeping gene GAPDH was used as an internal control for normalization of mRNA levels. cDNA was prepared as described. Amplification of TGF-ß1, CCL11, and GAPDH was performed using the primers pairs listed in Table 1. The probe for real-time PCR detection of CCL11 was 5'FAM-CCAGGGGCTCGCTGGGCCAG-BHQ-1; for TGF-ß1, 5'FAM-CCGCAAGGACCTCGGCTGGAAGTGGAT-BHQ-1; and for GAPDH, 5'HEX-CAAGCTTCCCGTTCTCAGCC-BHQ-1 (Integrated DNA Technologies, Inc., Coralville, IA). PCR reactions consisted of iQ Supermix (Bio-Rad), fibroblast cDNA, 200 µM dNTPs, and 200 µM of each primer and probe. PCR reactions went through 40 cycles of 30 s each at 95°C and 60°C in a Bio-Rad iCycler thermocycler with real-time detection occurring at 60°C.

RNase Protection Assay
The RNase protection assay was used to quantify mRNA in tissue samples. Total RNA was extracted from the fibroblast cells as described. For one set of reactions, single-stranded antisense RNA probes specific for phospholipase A2 (PLA2), cyclooxygenase (COX)-1, COX-2, 5-lipoxygenase (5-LO), 5-LO–activating protein (FLAP), and leukotriene C4 synthase, which are all of unique size, were synthesized and internally labeled with {alpha}32P-UTP. The plasmid necessary for generating these antisense probes was produced for this study through a contract with Pharmingen (San Diego, CA). A second plasmid set (hCK-3; Pharmingen) contained single-stranded antisense RNA probes for tumor necrosis factor (TNF)-ß, LTß, TNF-{alpha}, IFN-{gamma}, IFN-ß, TGF-ß1, TGF-ß2, and TGF-ß3. Probe and RNA preparations were allowed to hybridize overnight, after which samples were exposed to the RNase A and T1 for 1 h. Samples were washed, denatured, and electrophoresed on a polyacrylamide urea denaturing gel. Protected probes of an appropriate size were identified by comparison to a labeled ladder and quantified using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Data were normalized to the expression of the housekeeping probe present in the plasmid (L32).

Flow Cytometry
Cell-surface expression of CysLT1 and CysLT2 receptors on fibroblasts was evaluated by washing cell pellets with phosphate-buffered saline (PBS) supplemented with 5% donkey serum. Primary staining involved incubating fibroblasts with a 1:1,000 dilution of either CysLT1 or CysLT2 receptor antibody (Cayman, Ann Arbor, MI) followed by a secondary staining of a 1:10 dilution of a R-phycoerythrin–conjugated donkey anti-rabbit antibody (Abcam LTD, Cambridge, UK). For staining for the CysLT1 receptor, fibroblasts were first permeabilized using a commercial kit (Fix and Perm; Caltag Laboratories, Burlingame, CA) according to the manufacturer's instructions. Fluorescence was analyzed on a FAC-Scalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software. Data were analyzed as both the percentage of cells expressing each marker and as mean fluorescent intensity.

Cytoplasmic [Ca+2]i Assay
Fibroblasts were stimulated using 10 µM of LTC4, LTD4, prostaglandin (PG) 2F-{alpha}, sphingosine 1-phosphate, or lysophosphatidic acid (LPA). After starvation in serum-free medium for 12–24 h, cells were harvested and loaded with 1 µM Indo-1 AM in PBS for 30 min at 37°C. Cells were washed in PBS and resuspended at 2 x 106 cells/ml in a [Ca+2]i assay buffer (140 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 25 mM HEPES, and 10 mM glucose, pH 7.4). Cytoplasmic [Ca+2]i was determined at an extinction wavelength of 331 nm and an emission wavelength of 410 nm using a fluorescence spectrophotometer (Hitachi, F-4000; Gaithersburg, MD).

Statistical Analyses
Expression of mRNA transcripts was quantified using the phosphoimager and normalized to expression of the housekeeping gene L32. Cytokine, chemokine, and fibroblast secretion was quantified by ELISA. Data were contrasted between unstimulated fibroblasts and those stimulated with IL-4 using ANOVA for parametric data using JMP 3.2 software (Cary, NC) on a Macintosh G4 computer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4 Receptor Expression Fibroblasts
These studies were designed to evaluate the role of IL-4 in modulating cytokine, chemokine, fibronectin, and CysLT production from fibroblasts derived from nasal polyp tissue. Preliminary studies were performed to document the expression of IL-4 receptors on fibroblasts. We performed RT-PCR to document expression of the IL-4R{alpha} chain. All fibroblast cell lines expressed the IL-4R (a representative RT-PCR analysis is displayed in Figure 1A).




View larger version (122K):
[in this window]
[in a new window]
  		Figure 1. (A) IL-4R{alpha} mRNA expression from polyp fibroblast. Total RNA was extracted from polyp tissue and reverse-transcribed to make cDNA. PCR was performed using primers to amplify either the housekeeping gene GAPDH (lanes 2 and 4) or IL-4R{alpha} (lanes 3 and 5) and the products analyzed on 1.5% agarose gel. Lane 1 is a DNA ladder to mark the size of the PCR products. The gel displays representative PCR products from RNA derived from fibroblasts of two polyp patients. (B) Edg/LPA receptor expression on polyp fibroblasts. PCR was performed using primers to amplify either the housekeeping gene GAPDH (lanes 2 and 7) LPA1 (lanes 3 and 7), LPA2 (lanes 4 and 8), or LPA3 (lanes 5 and 9) and the products analyzed on 1.5% agarose gel. Lane 1 is a DNA ladder to mark the size of the PCR products.

 
CysLT Receptor Expression by Fibroblasts
We hypothesized that CysLT may directly contribute to the fibrosis and remodeling mediated by fibroblasts that is characteristic of CHES/NP. The fibroblast cell lines were therefore examined for expression of CysLT1 and CysLT2 receptors. In contrast to the IL-4R, neither of the CysLT receptors could be detected by RT-PCR (data not shown). By flow cytometry, we did not observe expression of CysLT1 receptors on fibroblasts; however, we were able to detect low levels of CysLT2 receptor expression on a small percentage of fibroblasts (10.5 ± 3.43%). Neither CysLT1 nor CysLT2 receptors could be induced when either IL-4 or IL-13 was added at 10 ng/ml to the cell cultures (data not shown). We evaluated the presence of functional CysLT receptors, including a putative CysLT3 receptor, by investigating the ability of CysLT to induce intracellular calcium fluxes. No Ca+2 shifts were observed when either LTC4 or LTD4 was added to cell cultures (data not shown). In contrast, however, these cells did respond to other proinflammatory lipid stimuli as calcium shifts were observed when PG2F-{alpha}, sphingosine 1-phosphate, or LPA was added to the cell cultures (data not shown). LPA signals through a subfamily of the endothelial differentiation gene receptor (edg) family (24). We investigated which LPA receptors were expressed on fibroblasts, and found that only LPA1 is constitutively expressed but that LPA2 receptors were induced upon stimulation with IL-4 (Figure 1B).

IL-4 Induction of Cytokine and Chemokine mRNA Transcript Expression by Fibroblasts
We evaluated the ability of IL-4 to induce expression of mRNA transcripts of cytokines and chemokines, which may contribute to the recruitment and activation of eosinophils and macrophages characteristic of CHES. Real-time PCR was performed (n = 10) for quantitation of CCL11 (eotaxin-1) and TGF-ß1 mRNA levels in unstimulated and IL-4–stimulated fibroblasts (a representative real-time PCR analysis for CCL11 is displayed in Figure 2). The mRNA expression (n = 15) for all other cytokines and chemokines tested was analyzed by semiquantitative agarose gel electrophoresis. The cumulative data for mRNA expression is presented in Table 2. TGF-ß1, IL-6, CCL11, and CCL13 (MCP-4) mRNA expression were upregulated when fibroblasts were stimulated with IL-4, whereas IL-11 mRNA expression was inhibited. No changes in mRNA levels were observed for GM-CSF, CCL5 (RANTES), or CXCL8 (IL-8).




View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Real-time PCR analysis of eotaxin expression. Fibroblasts were either left untreated or were stimulated with 10 ng/ml IL-4 for 24 h. Total RNA was extracted from polyp tissue and reverse-transcribed to make cDNA and real-time PCR was performed. Representative graphs from one fibroblast line showing upregulation of eotaxin mRNA expression with IL-4 stimulation (A) without upregulation of the housekeeping gene GAPDH (B). A: circles, eotaxin - IL-4; squares, eotaxin + IL-4. B: circles, GAPDH - IL-4; squares, GAPDH + IL-4.

 

View this table:
[in this window]
[in a new window]
  		TABLE 2 mRNA transcript expression in nasal polyp fibroblasts

 
RNase Protection Assay Analysis of Cytokine mRNA Expression in Polyp Fibroblasts
To confirm the increase in TGF-ß1 expression with IL-4 addition, a ribonuclease protection assay (RPA) was performed on unstimulated and IL-4–stimulated fibroblasts. A representative RPA assay from two fibroblast lines is displayed in Figure 3. As shown, the levels of both TGF-ß1 and TGF-ß2 are markedly increased in fibroblasts stimulated with IL-4. On longer exposures, small amounts of TGF-ß3 could be detected (data not shown) but no expression of IFN-{gamma}, IFN-ß, TNF-{alpha}, or TNF-ß could be observed in the unstimulated or IL-4–induced fibroblasts.



View larger version (56K):
[in this window]
[in a new window]
  		Figure 3. RNase protection assay of mRNA from IL-4–stimulated fibroblasts. Fibroblasts were either left untreated or were stimulated with 10 ng/ml IL-4 for 24 h. Total RNA was extracted from polyp tissue and allowed to hybridize with internally labeled {alpha}-32P–labeled uridine triphosphate single-stranded antisense RNA probes specific for TNF-{alpha}, TNF-ß, LTß, IFN-ß, IFN-{gamma}, TGF-ß1, TGF-ß2, TGF-ß3, L32, and GAPDH. Probes protected from RNase A and T1 digestion were electrophoresed on an acrylamide urea denaturing gel and identified. Representative studies from two subjects are shown. Lane 1 represents the undigested ladder, lanes 2 and 4 are unstimulated fibroblasts, and lanes 3 and 5 are fibroblasts stimulated with 10 ng/ml IL-4 for 24 h.

 
RPA Analysis of Metabolic Enzymes Involved in CysLT Synthesis
Even though CysLT receptors were not detected on the fibroblasts, it was possible that the fibroblasts could synthesize CysLT and contribute to the high levels observed in polyp tissue. Using an RPA probe to detect enzymes involved in arachidonic acid metabolism, COX-1 and LTA4 hydrolase were constitutively expressed in fibroblasts (data not shown). No stimulation was observed with IL-4 addition. 5-LO, FLAP, and LTC4 synthase were not expressed in resting fibroblasts and could not be induced by IL-4.

ELISA of Fibroblast Culture Supernatants
Fibroblasts from ten different patients were cultured either in the presence or absence of IL-4 and supernatants collected after 72 h for analysis of cytokine or chemokine expression (Table 3). Both IL-6 and CCL11 expression were significantly upregulated by IL-4, whereas CXCL8 and IL-11 displayed trends toward decreased expression that did not reach statistical significance. Cell surface fibronectin was insignificantly decreased upon IL-4 stimulation, with 1,233 ± 383 ng/ml fibronectin observed in the unstimulated fibroblasts compared with 682 ± 100 ng/ml in the IL-4–stimulated cells. TGF-ß1 protein levels were unchanged with IL-4 addition.


View this table:
[in this window]
[in a new window]
  		TABLE 3 Fibroblast cytokine and fibronectin protein production (ng/ml)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fibroblasts are an important component of polyp architecture that contribute collagen and fibronectin to the extracellular matrix. These studies were performed to evaluate both the ability of fibroblasts to contribute to the inflammatory milieu of CHES/NP and the role of IL-4 in activating these fibroblasts. We investigated both the production of proinflammatory cytokines and chemokines by fibroblasts and the ability of fibroblasts both to produce and respond to CysLTs that are critical to the development of this disorder. CHES/NP, especially when associated with aspirin intolerance, is associated with dramatic upregulation in CysLT production (22) and responsiveness (25) mediated, at least in part, through an upregulation of CysLT receptors on inflammatory cells (22). In addition to the remodeling and fibrosis that characterize both CHES and nasal polyp tissue, severe sinusitis has recently been associated with remodeling of the airway and diminished diffusing capacity (26). We therefore speculated that CysLT might function directly to activate fibroblast-mediated remodeling. However, our studies demonstrated that CysLT are not involved in fibroblast responses. These cells failed to express mRNA transcripts for either of the known CysLT receptors as shown by RT-PCR and, by flow cytometry, only a small subset of fibroblasts were shown to express low levels of the CysLT2 receptor. In addition, although IL-4 has been shown to upregulate expression of CysLT1 receptors on monocytes (20), it was unable to modulate expression of either class of receptor on nasal polyp–derived fibroblasts. These data extended to the related cytokine, IL-13, which is in contrast to the data of Chibana and coworkers (23), who demonstrated its ability to induce functional CysLT1 receptors on human lung fibroblasts. This contrast may reflect either inherent differences between lung- and nasal polyp–derived fibroblasts or the use by these investigators of a fetal lung tissue, which may have a unique phenotype. It is also plausible that both the cloning process, growth factors, and culture conditions used by these investigators may have modulated the phenotype of their fibroblasts, in contrast to our use of freshly-obtained nasal polyp tissue. The existence of a putative CysLT3 receptor has been suggested (27). LT receptors are all members of the G-protein–coupled receptor superfamily and are characterized by their ability to induce intracellular Ca+2 fluxes. We investigated the functionality of the CysLT2 receptor and the possible existence of such a third class of CysLT receptor through their ability to induce Ca+2 fluxes. The inability of either LTC4 or LTD4 to induce calcium fluxes strongly suggests the absence of functional CysLT receptors on nasal polyp–derived fibroblasts. In contrast, fibroblasts were activated by several lipid mediators, including PG2F{alpha}, sphingosine 1-phosphate, and LPA. LPA is an important proinflammatory mediator associated with allergic inflammation. We documented expression of one class of LPA receptors on fibroblasts (LPA1) and the induction by IL-4 of a second receptor (LPA2). Future studies are needed to address the importance of these mediators to polyp formation. In addition to not showing CysLT receptor expression on fibroblasts, these cells did not express any of the enzymes necessary for CysLT synthesis. Fibroblasts did express COX-1 and LTA4 hydrolase, suggesting their ability to produce both prostaglandins and LTB4. Previously, we have shown that polyps of patients with CHES have high concentrations of the CysLT and express the enzymes needed for CysLT synthesis (8). The results presented here suggest that other cells such as the eosinophil, mast cell, or mononuclear phagoyctes are the source of the CysLT found in the polyp tissue and not the fibroblast.

CHES is defined by the prominent presence of eosinophils (3, 4). We wanted to determine if fibroblasts could contribute mediators involved in eosinophil recruitment to the polyp. Stimulation of fibroblasts with IL-4 resulted in an increase of both mRNA production and protein secretion of CCL11 (eotaxin-1). Our results for IL-4–induced CCL11 expression from fibroblasts are in agreement with the results from Silvestri and colleagues, who also demonstrated that this could be inhibited by corticosteroids (28). Unlike in monocytes, where IL-4 decreases IL-6 expression (29), IL-4 significantly induced IL-6 expression from nasal fibroblasts (Table 3). Increased IL-6 expression may have direct effects on other cells by upregulating expression of other inflammatory mediators (30). This includes increasing levels of integrins and selectins that mediate local recruitment of inflammatory cells and synergy with other cytokines and products of the lipoxygenase pathway (31). A trend toward decreased levels of CXCL8 was observed in IL-4–stimulated fibroblasts. CXCL8 is a potent neutrophil chemoattractant (32), so decreased expression would be consistent with promotion of an eosinophilic infiltrate. Decreased IL-11 mRNA transcripts were consistently observed after IL-4 exposure along with a trend toward diminished IL-11 protein secretion. IL-11 has an important role of viral immune response, and diminished production of this cytokine (and perhaps CXCL8) may contribute to the increased severity and duration of upper respiratory viral infections in this disorder. An important component of the extracellular matrix in polyps is fibronectin, which is constitutively expressed in polyp-derived fibroblast cultures (Table 3). Surprisingly, although not statistically significant, addition of IL-4 resulted in a decrease in cell surface fibronectin. It is possible that IL-4 mediated release of fibronectin from the cell surface. Alternatively, diminished levels of fibronectin may reflect a change in cell physiology toward one that is more immunologically active. Further studies are needed to fully address this issue.

TGF-ß1 is produced by a variety of cells, including lymphocytes, eosinophils, macrophages, and fibroblasts, and exists as a latent protein composed of a mature dimer associated with latency-associated protein (LAP) (33). Activation occurs in vitro by heat or acidic pH disruption of the mature dimer and LAP, whereas in vivo, enzymatic action by glycosidases, proteases, and thrombospondin-1 release active TGF-ß1 from the latent complex (34). We observed a significant upregulation of TGF-ß1 mRNA production when fibroblast cultures were stimulated with IL-4; however, we did not observe an increase in secreted TGF-ß1 as measured by ELISA. It is possible that mRNA was increased without an increase in protein production. However, the ELISA kit that we used in these studies is designed to only detect active TGF-ß1 and not total (LAP-bound) TGF-ß1. It is therefore plausible that we may have failed to detect the increased TGF-ß1 as the extracellular factors required to activate the latent form of the protein were absent from our cultures. Our results demonstrating IL-4–inducible TGF-ß production by fibroblasts are consistent with recent studies that have shown that immunoreactive TGF-ß1 could be detected in nasal polyp tissue but not in tissue derived from normal nasal mucosa (35). In addition, our studies demonstrated basal expression of TGF-ß2 that was dramatically upregulated upon IL-4 stimulation. Our findings extend those of Eisma and coworkers, who previously demonstrated basal expression of both TGF-ß1 and TGF-ß2 in eosinophils present in polyp tissue (36). These results support a role for active TGF-ß1 and TGF-ß2 in stimulating proliferation of fibroblasts and facilitating nasal polyp growth.

Our concept regarding the linkage of CysLT overproduction and overresponsiveness in CHES/NP to airway remodeling/fibrosis is strongly supported in the LT literature. This has certainly been well argued in asthma, where CysLTs are associated with myofibroblast and smooth muscle proliferation and collagen deposition (37, 38) and leukotriene modifiers reduce airway remodeling (39). Similar to asthma, CysLTs are also thought to contribute to pulmonary fibrosis in both models of idiopathic pulmonary fibrosis (40) and in pulmonary scleroderma (41). CysLTs have similarly been associated with the fibrosis characteristic of hepatic cirrhosis (42) and systemic sclerosis (43). These data strongly support our concept that the overproduction of CysLTs in CHES/NP that we and others have reported could be directly related to the mechanisms of remodeling and polyp growth.

In conclusion, our results are supportive of a model in which fibroblasts contribute to the ongoing inflammatory processes involved in CHES and nasal polyposis and can create a state primed for continuous growth of existing polyps. The released proinflammatory and profibrotic factors have the potential act in an autocrine and paracrine manner. As has been suggested for the lung (44), we believe that the combination of lymphocytes, fibroblasts, and epithelial cells may function as a unit to mediate CHES and polyp formation. More work is required to understand the fine details of how these cells communicate during the early stages of disease to find new targets for intervention and treatment.


   Acknowledgments
 
This study was supported by NIH AI01793 and AI/HL47737, the American Lung Association AL440-ALA, and a medical school grant from Merck & Co., Inc.

Received in original form March 6, 2003

Received in final form August 6, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hamilos, D. L., D. Y. Leung, R. Wood, D. K. Bean, Y. L. Song, E. Schotman, and Q. Hamid. 1996. Eosinophil infiltration in nonallergic chronic hyperplastic sinusitis with nasal polyposis (CHS/NP) is associated with endothelial VCAM-1 upregulation and expression of TNF-{alpha}. Am. J. Respir. Cell Mol. Biol. 15:443–450.[Abstract]
  2. Borish, L. 2002. Sinusitis and asthma: entering the realm of scientific study. J. Allergy Clin. Immunol. 109:606–608.[CrossRef][Medline]
  3. Ferguson, B. J. 2000. Eosinophilic mucin rhinosinusitis a distinct clinicopathological entity. Laryngoscope 110:799–813.[CrossRef][Medline]
  4. Hamilos, D. L. 2001. Noninfectious sinusitis. ACI International 13:27–32.
  5. Bachert, C., M. Wagenmann, U. Hauser, and C. Rudack. 1997. IL-5 synthesis is upregulated in human nasal polyp tissue. J. Allergy Clin. Immunol. 99:837–842.[CrossRef][Medline]
  6. Minshall, E. M., L. Cameron, F. Lavigne, D. Y. M. Leung, D. Hamilos, A. Garcia-Zepada, M. Rothenberg, A. D. Luster, and Q. Hamid. 1997. Eotaxin mRNA and protein expression in chronic sinusitis and allergen-induced nasal responses in seasonal allergic rhinitis. Am. J. Respir. Cell Mol. Biol. 17:683–690.[Abstract/Free Full Text]
  7. Hamilos, D. L., D. Y. M. Leung, D. P. Huston, A. Kamil, R. Wood, and Q. Hamid. 1998. GM-CSF, IL-5, and RANTES immunoreactivity and mRNA expression in chronic hyperplastic sinusitis with nasal polyposis. Clin. Exp. Allergy 28:1145–1152.[CrossRef][Medline]
  8. Steinke, J. W., D. Bradley, P. Arango, C. D. Crouse, H. Frierson, S. E. Kountakis, M. Kraft, and L. Borish. 2003. Cytseinyl leukotriene expression in chronic hyperplastic sinusitis-nasal polyposis: Importance to eosinophilia and asthma. J. Allergy Clin. Immunol. 111:342–349.[CrossRef][Medline]
  9. Virchow, J. C., Jr., S. Faehndrich, C. Nassenstein, S. Bock, H. Matthys, and W. Luttmann. 2001. Effect of a specific cysteinyl leukotriene-receptor 1-antagonist (montelukast) on the transmigration of eosinophils across human umbilical vein endothelial cells. Clin. Exp. Allergy 31:836–844.[CrossRef][Medline]
  10. Ohshima, N., H. Nagase, T. Koshino, M. Miyamasu, M. Yamaguchi, K. Hirai, K. Yamamoto, N. Fujisawa, K. Kishikawa, and Y. Morita. 2002. A functional study on CysLT1 receptors in human eosinophils. Int. Arch. Allergy Immunol. 129:67–75.[CrossRef][Medline]
  11. Wang, Q. P., E. Escudier, F. Roudot-Thoraval, I. A. A. Samad, R. Peynegre, and A. Coste. 1997. Myofibroblast accumulation induced by transforming growth factor-ß is involved in the pathogenesis of nasal polyps. Laryngoscope 107:926–931.[CrossRef][Medline]
  12. Smith, S. J., A. M. Piliponsky, M. Hartman, and F. Levi-Schaffer. 2002. Stem cell factor: A possible paradigm between mast cells, eosinophils, and fibroblasts. ACI International 14:52–59.
  13. Doucet, C., D. Brouty-Boye, C. Pottin-Clemenceau, C. Jasmin, G. W. Canonica, and B. Azzarone. 1998. IL-4 and IL-13 specifically increase adhesion molecule and inflammatory cytokine expression in human lung fibroblasts. Int. Immunol. 10:1421–1433.[Abstract/Free Full Text]
  14. Moser, R., J. Fehr, and P. L. Bruijnzeel. 1992. IL-4 controls the selective endothelium-driven transmigration of eosinophils from allergic individuals. J. Immunol. 149:1432–1438.[Abstract]
  15. Schleimer, R. P., S. A. Sterbinsky, J. Kaiser, C. A. Bickel, D. A. Klunk, K. Tomioka, W. Newman, F. W. Luscinskas, M. A. Gimbrone, and B. W. McIntyre. 1992. Interleukin-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium: association with expression of VCAM-1. J. Immunol. 148:1086–1092.[Abstract]
  16. Hoontrakoon, R., J. Kailey, and D. Bratton. 1999. IL-4 and TNF-alpha synergize to enhance eosinophil survival. J. Allergy Clin. Immunol. 103:A239. (Abstr.)[CrossRef]
  17. Hsieh, C. S., A. B. Heimberger, J. S. Gold, A. O'Garra, and K. M. Murphy. 1992. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an alpha beta T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065–6069.[Abstract/Free Full Text]
  18. Seder, R. A., W. E. Paul, M. M. Davis, and B. Fazekas de St. Groth. 1992. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice. J. Exp. Med. 176:1091–1098.[Abstract/Free Full Text]
  19. Jutel, M., W. J. Pichler, D. Skrbic, A. Urwyler, C. Dahinden, and U. R. Muller. 1995. Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-gamma secretion in allergen-stimulated T cell cultures. J. Immunol. 154:4187–4194.[Abstract]
  20. Thivierge, M., J. Stankova, and M. Rola-Pleszczynski. 2001. IL-13 and IL-4 up-regulate cysteinyl leukotriene 1 receptor expression in human monocytes and macrophages. J. Immunol. 167:2855–2860.[Abstract/Free Full Text]
  21. Hsieh, F. H., B. K. Lam, J. F. Penrose, K. F. Austen, and J. A. Boyce. 2001. T helper cell type 2 cytokines coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by human cord blood-derived mast cells: profound induction of leukotriene C4 synthase expression by interleukin 4. J. Exp. Med. 193:123–133.[Abstract/Free Full Text]
  22. Sousa, A. R., A. Parikh, G. Scadding, C. J. Corrigan, and T. H. Lee. 2002. Leukotriene-receptor expression on nasal mucosal inflammatory cells in asprin-sensitive rhinosinusitis. N. Engl. J. Med. 347:1493–1499.[Abstract/Free Full Text]
  23. Chibana, K., Y. Ishii, T. Asakura, and T. Fukuda. 2003. Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin. J. Immunol. 170:4290–4295.[Abstract/Free Full Text]
  24. Fukushima, N., and J. Chun. 2001. The LPA receptors. Prostaglandins Other Lipid Mediat. 64:21–32.[CrossRef][Medline]
  25. Arm, J. P., S. O'Hickey, B. W. Spur, and T. H. Lee. 1989. Airway responsiveness to histamine and leukotriene E4 in subjects with aspirin-induced asthma. Am. Rev. Respir. Dis. 140:148–153.[Medline]
  26. ten Brinke, A., D. C. Grootendorst, J. T. Schmidt, F. T. de Bruine, M. A. van Buchem, P. J. Sterk, K. F. Rabe, and E. H. Bel. 2002. Chronic sinusitis in severe asthma is related to sputum eosinophilia. J. Allergy Clin. Immunol. 109:621–626.[CrossRef][Medline]
  27. Mellor, E. A., A. Maekawa, K. F. Austen, and J. A. Boyce. 2001. Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells. Proc. Natl. Acad. Sci. USA 98:7964–7969.[Abstract/Free Full Text]
  28. Silvestri, M., F. Sabatini, L. Scarso, A. Cordone, G. Dasic, and G. A. Rossi. 2002. Fluticasone propionate downregulates nasal fibroblast functions involved in airway inflammation and remodeling. Int. Arch. Allergy Immunol. 128:51–58.[CrossRef][Medline]
  29. Fenton, M. J., J. A. Buras, and R. P. Donnelly. 1992. IL-4 reciprocally regulates IL-1 and IL-1 receptor antagonist expression in human monocytes. J. Immunol. 149:1283–1288.[Abstract]
  30. Borish, L., and J. W. Steinke. 2003. Cytokines and chemokines. J. Allergy Clin. Immunol. 111:S460–S475.[CrossRef][Medline]
  31. Serhan, C. N., J. Z. Haeggstrom, and C. C. Leslie. 1996. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J. 10:1147–1158.[Abstract]
  32. Roebuck, K. A. 1999. Regulation of interleukin-8 gene expression. J. Interferon Cytokine Res. 19:429–438.[CrossRef][Medline]
  33. Letterio, J. J., and A. B. Roberts. 1998. Regulation of immune repsonses by TGF-ß. Annu. Rev. Immunol. 16:137–161.[CrossRef][Medline]
  34. Gleizes, P. E., J. S. Munger, I. Nunes, J. G. Harpel, R. Mazzieri, I. Noguera, and D. B. Rifkin. 1997. TGF-beta latency: biological significance and mechanisms of activation. Stem Cells 15:190–197.[Abstract/Free Full Text]
  35. Hirschberg, A., A. Jokuti, Z. Darvas, K. Almay, G. Repassy, and A. Falus. 2003. The pathogenesis of nasal polyposis by immunoglobulin E and interluekin-5 is completed by transforming growth factor-ß1. Laryngoscope 113:120–124.[CrossRef][Medline]
  36. Eisma, R. J., J. S. Allen, D. Lafreniere, G. Leonard, and D. L. Kreutzer. 1997. Eosinophil expression of transforming growth factor-beta and its receptors in nasal polyposis: role of the cytokines in this disease process. Am. J. Otolaryngol. 18:405–411.[CrossRef][Medline]
  37. Panettieri, R. A., E. M. L. Tan, V. Ciocca, M. A. Luttmann, T. B. Leonard, and D. W. P. Hay. 1998. Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction in vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am. J. Respir. Cell Mol. Biol. 19:453–461.[Abstract/Free Full Text]
  38. Medina, L., J. Perez-Ramos, R. Ramirez, M. Selman, and A. Pardo. 1994. Leukotriene C4 upregulates collagenase expression and synthesis in human lung fibroblasts. Biochim. Biophys. Acta 1224:168–174.[Medline]
  39. Henderson, W. R., L. O. Tang, S. J. Chu, S. M. Tsao, G. K. Chiang, F. Jones, M. Jonas, C. Pae, H. Wang, and E. Y. Chi. 2002. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am. J. Respir. Crit. Care Med. 165:108–116.[Abstract/Free Full Text]
  40. Wilborn, J., M. Bailie, M. Coffey, M. Burdick, R. Strieter, and M. Peters-Golden. 1996. Constitutive activation of 5-lipoxygenase in the lungs of patients with idiopathic pulmonary fibrosis. J. Clin. Invest. 97:1827–1836.[Medline]
  41. Kowal-Bielecka, O., O. Distler, K. Kowal, Z. Siergiejko, J. Chwiecko, A. Sulik, R. E. Gay, A. B. Lukaszyk, S. Gay, and S. Sierakowski. 2003. Elevated levels of leukotriene B4 and leukotriene E4 in bronchoalveolar lavage fluid from patients with scleroderma lung disease. Arthritis Rheum. 48:1639–1646.[CrossRef][Medline]
  42. Titos, E., J. Claria, R. Bataller, M. Bosch-Marce, P. Gines, W. Jimenez, V. Arroyo, F. Rivera, and J. Rodes. 2000. Hepatocyte-derived cysteinyl leukotrienes modulate vascular tone in experimental cirrhosis. Gastroenterology 119:794–805.[CrossRef][Medline]
  43. Kowal-Bielecka, O., O. Distler, M. Neidhart, P. Kunzler, J. Rethage, M. Nawrath, A. Carossino, T. Pap, U. Muller-Lander, B. A. Michel, S. Sierakowski, M. Matucci-Cerinic, R. E. Gay, and S. Gay. 2001. Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis. Arthritis Rheum. 44:1865–1875.[CrossRef][Medline]
  44. Davies, D. E., J. Wicks, R. M. Powell, S. M. Puddicombe, and S. T. Holgate. 2003. Airway remodeling in asthma: new insights. J. Allergy Clin. Immunol. 111:215–225.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
S. B. Early, E. Barekzi, J. Negri, K. Hise, L. Borish, and J. W. Steinke
Concordant Modulation of Cysteinyl Leukotriene Receptor Expression by IL-4 and IFN-{gamma} on Peripheral Immune Cells
Am. J. Respir. Cell Mol. Biol., June 1, 2007; 36(6): 715 - 720.
[Abstract] [Full Text] [PDF]

Home page
 Annals of Clinical & Laboratory ScienceHome page
G. Riccioni, C. Di Ilio, P. Conti, T. C. Theoharides, and N. D'Orazio
Advances in Therapy with Antileukotriene Drugs
Ann. Clin. Lab. Sci., October 1, 2004; 34(4): 379 - 387.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0071OCv1
30/2/212    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 Steinke, J. W.
Right arrow Articles by Borish, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Steinke, J. W.
Right arrow Articles by Borish, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.