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Published ahead of print on February 5, 2004, doi:10.1165/rcmb.2003-0380OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 92-99, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0380OC

Original Article

Pro- and Anti-Inflammatory Factors Cooperate to Control Hyaluronan Synthesis in Lung Fibroblasts

Thomas S. Wilkinson, Susan Potter-Perigo, Christina Tsoi, Leonard C. Altman and Thomas N. Wight

Department of Vascular Biology, Hope Heart Institute, Seattle; and Department of Medicine, Division of Allergy and Infectious Disease, and Department of Pathology, University of Washington, Seattle, Washington

Address correspondence to: T. N. Wight, Ph.D., Department of Vascular Biology, The Hope Heart Institute, 1124 Columbia Street, Seattle, WA 98104-2046. E-mail: twight{at}hopeheart.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyaluronan (HA) is an important constituent of the extracellular matrix and accumulates during inflammatory lung diseases like asthma. Little is known about the factors that regulate HA synthesis by lung cells. Accordingly, we investigated the effect of T-helper 1 (TH1) and 2 (TH2) cytokines and the anti-inflammatory agents fluticasone and salmeterol on HA synthesis in human lung fibroblasts. Interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF)-{alpha} were the most potent stimulators of HA synthesis and when combined, caused synergistic increases in HA accumulation. Time-course analysis of HA accumulation and [3H]-glucosamine incorporation into HA demonstrated continued synthesis over the 24 h of stimulation. Peak synthesis at 6–12 h coincided with an increased proportion of high molecular weight HA. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that IL-1ß and TNF-{alpha} induced HA synthase-2 messenger RNA (mRNA) 3 h following stimulation and remained elevated throughout the 24-h stimulation period. Fluticasone inhibited IL-1ß and TNF-{alpha} induced HA synthesis (44.5%) whereas salmeterol had no effect. When combined, fluticasone and salmeterol inhibited HA synthesis to a greater extent (85.2%). Further, fluticasone attenuated IL-1ß and TNF-{alpha} stimulated hyaluronan synthase-2 messenger RNA (mRNA), and the addition of salmeterol cooperatively enhanced this inhibition. These results indicate that enhanced synthesis of HA by the proinflammatory cytokines IL-1ß and TNF-{alpha} can be abrogated by specific corticosteroid and ß2 blocker combinations shown to be effective in the treatment of asthma.

Abbreviations: biotinylated proteoglycan, bPG • bovine serum albumin, BSA • complimentary DNA, cDNA • enzyme-linked immunosorbent assay, ELISA • extracellular matrix, ECM • ethylenediaminetetraacetic acid, EDTA • fetal bovine serum, FBS • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • high molecular weight, HMW • hyaluronan, HA • hyaluronan synthase, HAS • Interferon, IFN • interleukin, IL • messenger RNA, mRNA • nuclear factor {kappa}B, NF-{kappa}B • reverse transcriptase polymerase chain reaction, RT-PCR • T-helper type 1, TH1 • T-helper type 2, TH2 • tumor necrosis factor {alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is characterized by airway obstruction, increased hypersensitivity to inhaled allergens, and an abnormal accumulation of inflammatory cells, such as eosinophils, lymphocytes, mast cells, macrophages, and myofibroblasts (1–6). Airway remodeling is another important characteristic of asthma. Remodeling describes the structural alterations that occur in the lung due to prolonged chronic inflammation within the airways, and involves an abnormal accumulation of extracellular matrix (ECM) in the pulmonary epithelium and submucosa. Consequently, this imbalance in ECM affects airway resistance, compliance, and elasticity, leading to fibrosis and eventual loss of lung function (1–6). Current combination treatment with the glucocorticoid and long-acting ß2 agonist, fluticasone, and salmeterol, respectively, has proven clinically successful (7–9), but there is little evidence that these medications reverse remodeling events (6, 10).

Hyaluronan (HA) is a pleiotropic glycosaminoglycan residing in the ECM, composed of repeating disaccharide units of N-acetyl-D-glucosamine-ß (1->4)-D-glucuronic acid-ß (1->3). Polymerization of HA takes place at the plasma membrane by one or more of the three hyaluronan synthases (termed HAS 1, 2, and 3), which have distinct enzymatic properties (11–13). Accumulating evidence suggests that HA contributes to both lung homeostasis and disease. For instance, HA plays a role in the healthy lung by stimulating ciliary clearance, retaining homeostatic enzymes at the apical surface, and tethering and stabilizing lung surfactant molecules (14, 15). However, other studies suggest a role for HA during lung diseases. For example, the accumulation of fibrotic ECM components, in both animal models of lung fibrosis (16–19) and in milder forms of asthma in humans (20), is preceded by a rise in HA. HA also plays a major role in experimental lung inflammation, as CD44 (one of the major HA receptors) has been shown to be essential for the resolution of bleomycin-induced lung fibrosis (19). Further, intermediate weight HA, also termed HA fragments (< 2 x 105 Da), synergize with cytokines to upregulate chemokines in murine alveolar macrophages (21, 22). HA fragments can also affect ECM turnover in alveolar murine macrophages (23, 24). These studies suggest that understanding factors that produce HA during inflammation and disease is essential to designing targeted therapies limiting its negative consequences.

Asthmatic lungs contain increased levels of both the T-helper 1 (TH1) cytokines interleukin-1ß (IL-1ß), tumor necrosis factor (TNF)-{alpha}, and interferon-{gamma} (IFN-{gamma}) and the T-helper 2 (TH2) cytokines IL-4, IL-5, and IL-13 (25–27). These cytokines can affect ECM turnover and interactions. Specifically, IL-1ß, TNF-{alpha}, and IFN-{gamma} alone or in combination can differentially regulate proteoglycan production (28), induce matrix metalloproteinase (MMP)-3 (29), MMP-9, (30), and collagen I and III in resident lung cells (31). IL-4 and IL-13 alone or in combination can regulate {alpha} (I) collagen mRNA expression (32) and differentiation of lung fibroblasts to myofibroblasts (33). IL-5 binds heparin/heparan sulfates, suggesting that cytokines not only stimulate ECM production but also bind to and are retained by ECM (34).

Less is known about the roles of TH1 and TH2 cytokines in regulating the synthesis and turnover of HA by lung cells. It is not known whether therapeutic agents such as fluticasone and salmeterol have an effect on the synthesis of this molecule. Therefore, in this study, we have screened TH1 and TH2 cytokines, associated with lung disease, to determine if specific cytokines differentially regulate HA synthesis in human lung fibroblasts. We demonstrate for the first time that both TH1 and TH2 cytokine combinations can induce HA synthesis and that IL-1ß and TNF-{alpha} in combination were both the major stimulator of HA and also increased HA size. Further, IL-1ß and TNF-{alpha} in combination specifically induced the expression of HAS-2 with no effect on HAS-3. Increased HAS-2 expression could be markedly reduced by fluticasone and the addition of salmeterol cooperatively enhanced this effect. The results contained in this manuscript implicate specific pro- and anti-inflammatory factors in the modulation of HA synthesis by human lung fibroblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Supplies
IL-1ß, TNF-{alpha}, IL-4, IL-5, and IL-13 were purchased from R&D Systems, Inc. (Minneapolis, MN). IFN-{gamma} was purchased from Biosource International (Camarillo, CA). Fluticasone proprionate and salmeterol xinfoate were supplied by GlaxoSmithKline (Research Triangle Park, NC). Streptomyces hyaluronidase was purchase from ICN Biomedical Inc. (Aurora, OH). Dulbecco's modified Eagle's medium (DMEM), sodium pyruvate, nonessential amino acids, GlutMAX-1, penicillin-streptomycin, and trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Invitrogen Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was from Irvine Scientific (Santa Ana, CA).

Hyaluronan standards were prepared from high molecular weight (HMW) HA isolated from human umbilical cords (Sigma, St Louis, MO). HA-BSA was prepared by dissolving 100 mg HMW HA in 0.2 M NaCl and adjusting the pH to 4.7. While maintaining the pH at 4.7, 100 mg bovine serum albumin (BSA) and 20 mg 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) were added and the solution incubated for 1 h. Then HA-BSA was dialyzed extensively against PBS. Biotinylated proteoglycan (bPG), a specific probe for HA, was prepared as previously described (35). Streptavidin-labeled peroxidase was from Sigma.

Cell Culture
Human lung fibroblasts derived from explants of the lung, following removal of both the pleura and parenchyma, were a generous gift from Professor Ganesh Raghu, Divison of Pulmonary and Critical Care Medicine, University of Washington, Seattle, and were isolated as described previously in accordance with approval from the institution's human subjects review committee (36). Human lung fibroblasts were maintained in DMEM high-glucose medium supplemented (per 500 ml) with 55 ml FBS, 5 ml sodium pyruvate (100 mM), 5 ml nonessential amino acids (10 mM), 5 ml GlutMAX-1 (43 mg/ml), and 5 ml penicillin-streptomycin (penicillin G sodium, 10,000 U/ml, and streptomycin, sulfate 10 mg/ml) at 37°C in 5% CO2. Cells were passaged with trypsin-EDTA (0.05% trypsin and 0.53 mM tetrasodium EDTA) and were used for experiments between passages 5 and 11 after initial isolation.

Cytokine Stimulation
Human lung fibroblasts were seeded at 5 x 104/well in 24-well plates in 10% FBS DMEM. Following 24 h, cells were growth arrested for 48 h in 0.1% FBS DMEM at which point the cells were ~ 80% confluent. Medium was then removed and cells were stimulated with cytokines for 24 h in fresh 0.1% FBS DMEM. The media were assessed for HA levels and the cell layer for total DNA content.

To assess HAS expression, human lung fibroblasts were seeded at 5 x 105/60 mm dishes and the protocol for cytokine stimulation performed. At specific time points (1, 3, 6, 12, and 24 h) following stimulation, total RNA was isolated as described below. Finally, to assess constitutive HAS expression, human lung fibroblasts were seeded at 5 x 105/60 mm dishes and allowed to grow under normal culture conditions in 10% FBS DMEM for 48 h before RNA isolation.

HA Enzyme-Linked Immunosorbent Assay
This is a competitive enzyme-linked immunosorbent assay (ELISA) in which the samples to be assayed were first mixed with bPG and then added to an HA-coated microtiter plate, the final signal being inversely proportional to the level of HA. To isolate samples, medium from cultures was digested with papain (5 mg/ml) in 0.1 M Tris/acetate, 5 mM EDTA, 5 mM L-cysteine hydrochloride (HCl) at pH 7.3 for 18 h at 60°C. Following digestion, the papain was inactivated by heating to 100°C for 20 min. For ELISA, we used a modification of that previously described (37). Briefly, on Day 1, 75 µl of standards and samples were incubated with 75 µl bPG (5 µg/ml) overnight. Also on Day 1, 100 µl of HA-BSA preparation was added to each well of a Nunc-Immuno 96-well plate (Nalge Nunc International) and incubated for 1 h at room temperature. Plates were washed x3 in PBS using a Denley Wellwash 4 mechanical plate washer. Then, 200 µl of 10% calf serum (CS) in PBS was added and incubated overnight. On Day 2, plates were washed as before and then 60 µl of the incubated bPG/HA standards and samples were added to duplicate wells and incubated for 2 h at room temperature. Plates were washed as before and then 60 µl of streptavidin-labeled peroxidase (2 µg/ml) was added to each well and incubated for 30 min at room temperature. Plates were washed as before and then 60 µl of 2,2' azinobis 3-ethyl-benzthiozoline sulfonic acid (AEBT-SA) in 0.1 M sodium citrate pH 4.2 was added. The resulting absorbances were measured at 405/570 nm on a OPTImax microplate reader (Molecular Devices, Sunnyvale, CA) using SOFTmax PRO (version 4.3) software (Molecular Devices). Readings at 570 nm were subtracted from those at 405 nm to account for plate imperfections.

HA Synthesis
Human lung fibroblasts were seeded at 1.7 x 105/well in 6-well plates in 10% FBS DMEM. Following 24 h, cells were growth arrested for 48 h in 0.1% FBS DMEM and then stimulated with 1 ng/ml of both IL-1ß and TNF-{alpha}. To assess temporal changes in HA synthesis, 25 µCi/ml of [3H]-glucosamine was added at 6 h intervals over the next 36 h. Supernatants and cell layers were isolated and digested with papain (5 mg/ml) in 0.1 M Tris/acetate, 5 mM EDTA, 5 mM L-cysteine hydrochloride at pH 7.3 for 18 h at 60°C. Following digestion, the papain was inactivated by heating to 100°C for 20 min. Radiolabeled macromolecules were then recovered and separated from unincorporated precursor by precipitation on nitrocellulose membranes using slot blot analysis. Briefly 250 µl of sample was added to an equal volume of 2% cetylpyridinium chloride (CPC), 50 mM NaCl buffer and the solution blotted onto nitrocellulose membrane. The membrane was washed six times in 2% CPC, 50 mM NaCl buffer and once in deionized water before air-drying at room temperature overnight. Incorporation of [3H]-glucosamine into HA was measured by digesting an equivalent radiolabeled aliquot with Streptomyces hyaluronidase (2 U/ml) for 24 h at 37°C, before slot blotting. HA was measured as the amount of hyaluronidase-sensitive material precipitated to the nitrocellulose membrane.

HA Size Comparison
The relative molecular size of the HA synthesized by control and IL-1ß- and TNF-{alpha}–treated cells was compared by chromatography of [3H]-glucosamine-labeled glycosaminoglycans on a 1.2 x 58-cm column of Sephacryl S-1000 eluted in 0.5 M sodium acetate, 0.025% Chaps (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 0.02% sodium azide at pH 7.0. An equivalent aliquot was digested with Streptomyces hyaluronidase before chromatography to identify radiolabeled HA. A blue dextran calibration standard with a molecular weight of 2 x 106 Da was used for comparison.

DNA Assay
Total cellular DNA was assayed as previously described (38). Briefly, cells were washed very gently in PBS and then frozen at –20°C until the day of assay. After this, the cells were allowed to reach room temperature and 2 ml of 10 mM EDTA (pH 12.3) was added and the cells incubated for 20 min at 37°C. Samples were then neutralized to pH 7.0 with 1 M KH2PO4. Salmon sperm DNA was used as a standard and was treated similarly. To measure DNA concentration, 1 ml of Hoescht 33,258 dye (200 ng/ml) was added to 1 ml of DNA and fluorescence measured with excitation and emission wavelengths of 350 and 455 nm respectively on a Turner Quantech Digital Filter Fluorometer, Model FM 109,515 (Barnstead/Thermolyne, Dubuque, IA).

RNA Isolation
Total RNA was isolated as previously described (39). In brief, cells were cultured in 60 mm dishes, the medium removed, and the plates placed on ice. Then 1.5 ml of cold TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) was added and the cell layers incubated on ice for 5 min after which 300 µl of chloroform/isoamyl alcohol (24:1) was added and the samples mixed and then centrifuged at 13,000 rpm for 20 min. A total of 700 µl of the aqueous phase was mixed with an equal volume of isopropanol, incubated at –70°C overnight, centrifuged at 13,000 rpm for 20 min before washing once in 100 µl of 75% ethanol and finally resuspended in 20 µl of sterile water. Finally, deoxyribonuclease (DNase) was added at 0.02 U/µl and the samples incubated at 37°C for 15 min. The RNA was purified by adding 100 µl of acid (pH 4.5) phenol:chloroform:isoamyl alcohol (25:24:1) to an equal volume of DNase-treated sample, mixed and centrifuged. Samples were extracted once again in chloroform:isoamyl alcohol (24:1) and precipitated using 80 µl RNA sample, 8 µl 3 M sodium acetate, and 200 µl of ethanol. Samples were finally washed in 100 µl of 70% ethanol and resuspended in sterile water. Total RNA was quantified by measuring the absorbance at 260 and 280 nm in a Beckman DU640 spectrophotometer (Fullerton, CA). The purity of the samples was always > A260/A280 = 1.8.

Reverse Transcriptase-Polymerase Chain Reaction
Expression of the three isoforms of HAS was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Synthesis of single-strand cDNA was performed in 50 µl reactions (reagents were purchased from Promega unless stated otherwise) using 1 µg total RNA, 2 µl oligo-dT (Ambion, Austin, TX), 2.5 µl RNase inhibitor (40 U/µl), 5 µl deoxynucleotide triphosphates (dNTP), 10 µl 5x Improm-II buffer, 12 µl MgCl2 (25 mM), and 2.5 µl Improm-II reverse transcriptase per RNA sample. Annealing was performed at 25°C for 5 min and cDNA single-strand synthesis was at 42°C for 90 min.

Conditions for PCR amplification were optimized for MgCl2 concentration and temperature using a PTC-200 peltier thermal cycler (MJ Research, Waltham, MA). PCR reactions were performed using 1 µl single-strand cDNA product, 250 µM dNTP, 2 µl 10x Amplitaq Gold buffer, 5 pM of each primer, 0.75 U Amplitaq Gold enzyme (Applied Biosystems, Foster City, CA), and MgCl2 (1.6 mM HAS-1, 1.6 mM HAS-2, 2.2 mM HAS-3, and 1.6 mM GAPDH). After a 10 min initial enzyme activation step at 95°C for the Amplitaq Gold, denaturation was done for 1 min at 95°C, annealing at 59.5°C for 1 min, and extension at 72°C for 1 min. Data on the specific primers used in this study are shown in Table 1.


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  		TABLE 1. Primer sequences

 
Statistical Analysis
One-way ANOVA was used to test if differences existed between treatment groups with a P < 0.05 being considered significant. To further analyze the specific differences between groups, we used a series of t tests with Bonferroni adjustment. All statistical tests were performed using Statistics Calculator, Version 8.0 software, 1997–2003 (StatPac Inc., Minneapolis, MN).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specific Cytokines Regulate HA Accumulation by Human Lung Fibroblasts
Increasing doses of the TH1 cytokines IL-1ß, TNF-{alpha}, and IFN-{gamma} and the TH2 cytokines IL-4, IL-5, and IL-13 were added to growth-arrested cultures of lung fibroblasts (Figures 1A and 1B, respectively). IL-1ß and TNF-{alpha} caused a dose-dependent increase in HA accumulation. Furthermore, IL-4 had a mild inhibitory effect on HA production. IL-5, IL-13, and IFN-{gamma} had no dose-dependent effects on HA accumulation. Further, cultures stimulated with 10% FBS had an equivalent HA accumulation to those stimulated with 10 ng/ml IL-1ß on a per DNA basis. Analysis of total cellular DNA revealed no significant difference between control and any of the cytokine-stimulated cultures, suggesting that under these conditions these cytokines do not induce cell proliferation (data not shown). Total cellular DNA isolated from cultures exposed to 10% FBS, however, was significantly increased suggesting that these cells had initiated the cell cycle.



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  		Figure 1. Effect of TH1 and TH2 cytokines on HA production by human lung fibroblasts. Human lung fibroblasts were seeded and made quiescent as in MATERIALS AND METHODS. Then, IL-1ß (black bar), TNF{alpha} (light gray bar), and IFN-{gamma} (medium gray bar) (A) or IL-4 (black bar), IL-5 (light gray bar), and IL-13 (medium gray bar) (B) were added in increasing doses (1–10,000 pg/ml) for 24 h. Supernatants were collected for determination of HA levels and cell layers were washed gently and then frozen at –20°C for total DNA measurement. Results are expressed as nanograms HA per micrograms DNA and are the mean ± SEM (n = 4) of data from one of three experiments. *P < 0.05, cytokine response > control. #P < 0.05, cytokine response < control. White bars, control.

 
To investigate synergistic interactions (Figure 2, gray bars), cytokines were combined and then added to growth arrested lung fibroblasts. Single cytokine responses were also included in this experiment (Figure 2, white bars, inset) and the results used to calculate expected additive values (Figure 2, black bars). The combinations IL-1ß/TNF-{alpha}, IL-1ß/IL-13, and IL-1ß/IFN-{gamma} resulted in significant synergistic increases in HA accumulation compared with predicted additive values. Similarly, the combinations IL-4/IL-5, IL-4/IL-13, and IL-5/IL-13 also resulted in significant synergistic increases although the absolute level of HA was smaller than the IL-1ß/TNF-{alpha}, IL-1ß/IL-13, and IL-1ß/IFN-{gamma} combinations. Conversely, the combinations TNF-{alpha}/IL-5 and TNF-{alpha}/IL-13 produced a negative synergism where HA production was significantly less than predicted additive values. Such results indicate both positive and negative regulation by TH1 and TH2 cytokines and suggest a variety of mechanisms for differentially regulating HA accumulation. Because the combination of 1 ng of IL-1ß and 1ng of TNF-{alpha} had the greatest effect on HA production of the screened cytokines, this combination was used for all subsequent experiments.



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  		Figure 2. Effect of dual cytokine stimulation on HA production by human lung fibroblasts. Human lung fibroblasts were seeded and made quiescent as in MATERIALS AND METHODS. Cytokines (1 ng/ml) were added either alone (inset, white bars) or in combination (gray bars) for 24 h. Supernatants were collected for determination of HA levels and cell layers were washed gently and then frozen at –20°C for total DNA measurement. The predicted HA response to dual cytokine stimulation (black bars) was calculated by adding the values of the two cytokines individually minus the control response (using inset values). Results are expressed as nanograms HA per µg DNA and are the mean ± SEM (n = 4) of data from one of two experiments. *P < 0.05, dual cytokine response > predicted additive value. #P < 0.05, dual cytokine response < predicted additive value. +P < 0.05 single cytokine > control.

 
Time-course analysis of HA accumulation during the first 24 h of stimulation demonstrated that, IL-1ß and TNF-{alpha}, HA continued to accumulate in the media over this period. As the proportion of HA secreted into the media increased with time and treatment (Figure 3, inset) we only analyzed media fractions 24 h after treatment in subsequent experiments. Analysis of de novo HA biosynthesis by [3H]-glucosamine incorporation, assessed during 6-h labeling periods, confirmed that IL-1ß and TNF-{alpha} stimulation increased HA synthesis compared with control, at time points between 6 and 24 h post-stimulus, reaching a peak at 6–12 h, suggesting that over the time course studied the specific activity of radiolabeled HA remained unchanged. Analysis of total cellular DNA revealed no significant difference between control and IL-1ß and TNF-{alpha} stimulated cultures, suggesting that under these conditions IL-1ß and TNF-{alpha} do not induce cell proliferation (data not shown).



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  		Figure 3. Effect of IL-1ß and TNF-{alpha} on the time course of HA accumulation by human lung fibroblasts. Human lung fibroblasts were seeded and made quiescent as in MATERIALS AND METHODS. Then IL-1ß and TNF-{alpha} were added for 24 h. At 6-h intervals, supernatants were collected for determination of HA levels in the media. The cell layers were washed gently and then frozen at –20°C for determination of HA in the cell layer. Cell layers in parallel wells were washed gently and then frozen at –20°C for total DNA measurement. Results are expressed as nanogram HA per microgram DNA and are the mean ± SEM (n = 4) of data from one of three experiments. White, hatched bars, control cell layer; gray hatched bars, IL-1ß + TNF{alpha} cell layer; white bars, control media; gray bars, IL-1ß + TNF{alpha} media. In the inset plot, the same data is presented as the percentage HA in the media. *P < 0.05 IL-1ß/TNF{alpha} versus time point control in the media. Inset: white circles, control; closed circles, IL-1ß + TNF{alpha}.

 
IL-1ß and TNF-{alpha} Influence the Relative Molecular Size of HA Polysaccharide
During the first 6 h of stimulation (Figure 4) there was no difference in the size distribution of HA molecules as measured by hyaluronidase sensitive [3H]-glucosamine–labeled macromolecular material whether treated with IL-1ß and TNF-{alpha} (black) or not (white). However, by 6–12 h, the proportion of HA above the 2 x 106 Da standard increased in the IL-1ß- and TNF-{alpha}–treated samples compared with control. HA labeled at 12–18 h also showed increases in the proportion of HMW HA, although the size difference appeared to be decreasing over this period until at 18–24 h there was no HA size difference between treatments. Analysis of cell layer fractions labeled in the same manner also produced similar results with increased molecular weight HA at 6–12 h that returned to its original size following 18–24 h. Interestingly, however, the differences observed in the cell layer were not as dramatic as those in the media. Further, in the cell layer there was no radioactive material in the void volume of the column.



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  		Figure 4. Effect of IL-1ß and TNF-{alpha} on HA size. Total incorporated [3H]-glucosamine-labeled glycosaminoglycan samples (isolated by G50 Sephadex chromatography) were used to determine the relative molecular size of HA synthesized by control and IL-1ß- and TNF-{alpha}–treated human lung fibroblasts, using Sephacryl S-1000 chromatography. Both Streptomyces hyaluronidase treated and nontreated samples (25,000 dpm) were added to Sephacryl S-1000 columns to identify the HA peak. Supernatants from 3–6 wells were pooled. Results are expressed as the distribution of hyaluronidase sensitive [3H]-glucosamine per fraction and are the mean ± SEM from three replicates. Results were used from fractions 23–46 (Kav ~ 0.6) where hyaluronidase sensitive material eluted. Column V0 and VT were at fractions 23 and 64, respectively. This is representative data from one of two experiments. The downward arrows indicate the position where the blue dextran standard (2 x 106 Da) eluted. White circles, control; black circles; IL-1ß and TNF-{alpha}.

 
IL-1ß and TNF-{alpha} Induce a Specific HAS Isoform
To determine if a specific HAS isoform is involved in IL-1ß and TNF-{alpha} induced HA synthesis levels of mRNA, transcripts for HAS-1, 2, and 3 were determined by RT-PCR. Specific transcript for HAS-2 and HAS-3 could be detected following 25 and 35 cycles respectively (Figure 5A). HAS-1 transcript could not be detected even after 40 cycles (Figure 5A). These results suggest that HAS-2 is the major isoform expressed in lung fibroblasts. Next, we assessed the effect of IL-1ß and TNF-{alpha} on HAS-2 and HAS-3 expression. Following IL-1ß and TNF-{alpha} treatment, HAS-2 mRNA was increased at 3 h, with the transcript remaining elevated up to 24 h post-stimulation (Figure 5B). At the same time, IL-1ß and TNF-{alpha} had no effect on HAS-3 (Figure 5B). This suggests that (i) HAS-2 is responsible for the prolonged de novo synthesis over the first 24 h of stimulation (Figure 3) and (ii) that HAS-2 might also be responsible for the increase in HMW HA between 6–12 h post-stimulation (Figure 4).



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  		Figure 5. Regulation of hyaluronan synthase expression in human lung fibroblasts. (A) Human lung fibroblasts were seeded and allowed to grow for 2 d in 10% FBS/DMEM. Then total RNA was isolated and purified as in MATERIALS AND METHODS. One microgram of total RNA was subjected to RT-PCR using specific primers for HAS-1, HAS-2, HAS-3, and GAPDH with increasing cycles of PCR to determine transcript levels. Results are presented as a representative gel from three experiments. (B) Human lung fibroblasts were plated and made quiescent as described in MATERIALS AND METHODS. IL-1ß and TNF-{alpha} (1 ng/ml each) were then added and total RNA isolated following 1, 3, 6, 12, and 24 h. One microgram of total RNA was subjected to RT-PCR using specific primers for HAS-2, HAS-3, and GAPDH (32, 37, and 28 cycles respectively) to determine transcript levels. Results are presented as a representative gel from three experiments.

 
Anti-Inflammatory Agents Influence the Synthesis of the Glycosaminoglycan HA by Influencing HAS-2 mRNA
Fluticasone and salmeterol have been shown to be a successful combination therapy in the treatment of asthma (7–9). We investigated the effects of these drugs alone and in combination on both constitutive and IL-1ß and TNF-{alpha} induced HA production by human lung fibroblasts (Figure 6). Fluticasone alone, in a dose-dependent manner, downregulated IL-1ß and TNF-{alpha} induced HA production at concentrations between 10–10 and 10–6 M (Figure 6, black circles). Fluticasone added alone had no effect on constitutive HA production (Figure 6, white circles). In contrast, salmeterol had no effect on IL-1ß and TNF-{alpha} induced HA production up to 10–6 M (Figure 6, black triangles). However, salmeterol attenuated constitutive HA production at concentrations from 10–10 to 10–6 M although values did not quite reach significance (white triangles). Furthermore, the combination of fluticasone and salmeterol produced 4-fold greater downregulation of IL-1ß and TNF-{alpha} induced HA production than produced by fluticasone alone (10–6 M, black squares versus circles). Also, when added together, fluticasone and salmeterol significantly attenuated constitutive HA production (white squares).



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  		Figure 6. Effect of fluticasone and salmeterol on constitutive and IL-1ß- and TNF-{alpha}–induced HA production. Human lung fibroblasts were seeded and made quiescent as described under MATERIALS AND METHODS. Then IL-1ß and TNF-{alpha} (1 ng/ml each) were added and fluticasone and salmeterol were included either alone or in combination for 24 h. Supernatants were collected for determination of HA levels and cell layers were washed gently and then frozen at –20°C for total DNA estimation. Results are expressed as nanogram HA per microgram DNA and are the mean ± SEM (n = 4) of data from one of two experiments. *P < 0.05, IL-1ß/TNF-{alpha} versus IL-1ß/TNF-{alpha}/fluticasone. #P < 0.05 IL-1ß/TNF-{alpha}/fluticasone versus IL-1ß/TNF-{alpha}/fluticasone/salmeterol. +P < 0.05 Control versus fluticasone/salmeterol alone. White circles, flucasone alone; white triangles, salmeterol alone; white squares, flucasone + salmeterol; Black circles, IL-1ß/TNF{alpha} + flucasone; black triangles, IL-1ß/TNF{alpha} + salmeterol; black squares, IL-1ß/TNF{alpha} + flucasone + salmeterol.

 
To investigate the mechanism behind this inhibition, we isolated total RNA from human lung fibroblasts treated with IL-1ß and TNF-{alpha} (1 ng/ml each) in the presence of fluticasone and salmeterol (10–8 M each) and then assessed the levels of HAS-2 mRNA (Figure 7). Neither fluticasone nor salmeterol when added alone or in combination had any effect on constitutive HAS-2 expression (Figure 7). Conversely, fluticasone significantly attenuated IL-1ß- and TNF-{alpha}–induced HAS-2 mRNA expression when added alone. Salmeterol when added alone had no effect on IL-1ß- and TNF-{alpha}–induced HAS-2 mRNA transcript levels, but when combined with fluticasone, further significantly downregulated the attenuated HAS-2 message produced by fluticasone alone.



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  		Figure 7. Effect of fluticasone and salmeterol on IL-1ß- and TNF-{alpha}–induced HAS-2 expression in human lung fibroblasts. Human lung fibroblasts were seeded and made quiescent as described in MATERIALS AND METHODS. Then IL-1ß and TNF-{alpha} (1 ng/ml each) were added and fluticasone and salmeterol (10–8 M each) were included either alone or in combination. Total RNA was isolated following 6 h and subjected to RT-PCR analysis using specific primers for HAS-2 and GAPDH (32 and 30 cycles, respectively). Results are expressed as the ratio of optical density values from HAS-2 and GAPDH bands and are the mean ± SEM (n = 3) of data from one of two experiments. The gel is representative from one of those experiments. *P < 0.05, Control versus IL-1ß/TNF-{alpha}. +P < 0.05, IL-1ß/TNF-{alpha} versus IL-1ß/TNF-{alpha}/fluticasone. #P < 0.05, IL-1ß/TNF-{alpha}/fluticasone versus IL-1ß/TNF-{alpha}/fluticasone/salmeterol.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this present study, IL-1ß and TNF-{alpha} were identified as important stimulators of HA production in adult human lung fibroblasts. Further, other novel cytokine combinations that induce HA synthesis were found. IL-1ß and TNF-{alpha} also increased HA size. In addition, we showed that the increased production of HA is consistent with induction of the HAS-2 isoform. Finally, we showed that two mainstays of asthma therapy, fluticasone and salmeterol, can inhibit both constitutive and IL-1ß– and TNF-{alpha}–induced HA production when given in combination and that this is consistent with the attenuation of HAS-2 mRNA.

When given alone, the TH1 cytokines IL-1ß, TNF-{alpha}, and IFN-{gamma} dramatically induced HA production, whereas the TH2 cytokines IL-4, IL-5, and IL-13 had no effect or were slightly inhibitory in the case of IL-4. Previous studies have shown that IL-1ß, TNF-{alpha}, and IFN-{gamma} can stimulate HA production in lung fibroblasts (28, 40). What was particularly striking in the present study was the synergistic interactions between these and other cytokines (Figure 2). It is likely that cells and tissues of the lung release multiple cytokines into the airways (25–27) suggesting a significant amount of crosstalk between signaling pathways that might activate transcription of the HAS isoenzymes. Recent analysis of the proximal promoter regions for the human HAS genes identified several potential transcription factor binding sites for the induction of HA by the inflammatory cytokines used in our study (41). Nuclear factor (NF)-{kappa}B is present in the upstream region of all HAS genes and might be responsible for the induction of HA by IL-1ß and TNF-{alpha} in lung fibroblasts as shown previously in MRC lung myofibroblasts (42). IL-13 synergizes positively with IL-1ß, suggesting a possible role for IL-1ß and IL-13 for the production of HA in the lung. Consistent with this is the fact that IL-13 transgenic mice with targeted expression to the lung, show increased levels of HA in bronchoalveolar lavage fluid (43). Our finding that IL-4, IL-5, and IL-13 can stimulate HA synthesis when combined (but not alone) is novel and suggests the cooperative crosstalk of their STAT signaling pathways (44) for the induction of HAS enzymes. In contrast, the TNF-{alpha}/IL-5 and the TNF-{alpha}/IL-13 combinations showed a negative synergism, suggesting the potential for inhibitory crosstalk mechanisms. Interestingly, STAT binding concensus sequences do not appear to be common among the HAS proximal promoters (41), suggesting the potential for these transcription factors to differentially regulate HAS isoforms. Further investigation of signaling pathways and the distal HAS promoters could increase our understanding of the factors that regulate HA synthesis.

IL-1ß and TNF-{alpha} not only influenced the total amount of HA produced but also influenced its molecular weight. HA molecules with various size distributions have the potential to produce different activities contributing to the amplification of both the inflammatory and remodeling responses within the lung. First, ECM enriched in HMW HA tends to favor proliferative responses (45) and so might induce the hyperplasia of smooth muscle cells characteristic of the remodeling response. Second, HMW HA is capable of forming intercellular cables, thereby producing an inflammatory matrix that supports leukocyte retention. Indeed, such a possibility has been demonstrated during inflammatory bowel disease (46, 47). Third, recent studies have suggested that intermediate size HA (0.2 x 106 Da) can induce a plethora of chemokines from alveolar macrophages (21, 22) including monokine induced by IFN-{gamma} (MIG), IFN-{gamma}–inducible protein-10 (IP-10), macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, cytokine responsive gene-2 (CRG-2), monocyte chemoattractant peptide-1 (MCP-1), regulated upon activation, normal T cells expressed and secreted, and mouse homolog of growth-related oncogene-{alpha}. These results demonstrate that HA has the potential to indirectly recruit leukocyte subsets to the alveolar space for development of the inflammatory response.

Prolonged accumulation of HA with a range of molecular weights could be responsible in part for the maintenance of the fibrotic response. Specifically, small HA or HA oligosaccharides (1–4 x 103 Da) have been shown to induce the expression of collagen I and III (48) in rat lung fibroblasts. In the bleomycin model of lung fibrosis, YKQKIKHVVKLK, a specific HA binding peptide, was able to attenuate macrophage motility and accumulation, total lung collagen–{alpha}-(I) mRNA and hydroxyproline content (17). These results suggest that HA has a role to play in both the initial and later phases of lung injury. Indeed a recent study by Teder and coworkers suggests that excessive accumulation of intermediate molecular weight HA (0.02–0.5 x 106 Da) and HMW HA (1–2 x 106 Da) is lethal, as CD44-deficient mice succumb to bleomycin-induced fibrosis and lung injury (19). The accumulation of intermediate molecular weight (< 1 x105 Da) HA might contribute to the increases in apoptotic neutrophils and attenuation of TGF-ß activity, so prolonging the intrapulmonary inflammation in this disease. Indeed, HA oligosaccharides (1.2–4 x 103 Da) have been shown to induce apoptosis, by upregulating caspase-3 (49), however, there are no data to suggest that HA inhibits apoptotic cell removal and so promotes accumulation of these cells.

Fluticasone and salmeterol have proved to be a very successful prophylactic treatment for asthma and bronchial hyper-responsivenes (7–9). To date, other studies have suggested that glucocorticoids such as dexamethasone (50) and hydrocortisone (13) can attenuate HAS-2 mRNA in dermal fibroblasts, MG-63 osteoblast-like cells, and mesothelial cells. More pertinent to pulmonary disease and asthma, however, is a study using lung fibroblasts, showing that the combination of fluticasone and salmeterol can downregulate intercellular adhesion molecule-1 (ICAM-1) and hyaluronic-acid adhesion molecule (HCAM, also termed CD44) expression, and further, that salmeterol could enhance the inhibitory effect of fluticasone on ICAM but not HCAM expression (51). The mechanism to attenuate HAS-2 mRNA likely involves the downregulation of NF-{kappa}B activity possibly by inducing I-kB (52, 53). Consistent with this is that the HAS-2 promoter contains cyclic AMP-responsive element binding protein (CREB) and NF-{kappa}B, two possible downstream targets of ß-agonists and glucocorticoids respectively (41). Our observation that HA levels decrease below control values when HAS-2 mRNA only decreases to control levels suggests HA turnover may be affected. However, to our knowledge, no studies to date have addressed the activity of fluticasone and salmeterol on the hyaluronidases. Interestingly, IL-1ß/TNF-{alpha} and TNF-{alpha}/IFN-{gamma} combinations respectively have been shown to decrease and increase hyaluronidase activity in human lung fibroblasts (40).

Inhibition of HA production by attenuating HAS-2 expression is one generally unrecognized mechanism which might contribute to the therapeutic action of fluticasone and salmeterol. This raises the question whether inhibiting HA production would be beneficial in the asthmatic airway. Inhibiting the synthesis of intermediate molecular weight (< 1 x 105 Da) HA could contribute to decreasing macrophage activation and leukocyte recruitment by a mechanism that involves a decrease in chemokine production. Inhibiting HA might also contribute to attenuating the production of a prefibrotic matrix and so decrease fibrosis and collagen deposition. Such a hypothesis is supported by the fact that HA induces collagen (48), and that levels of HA in BAL are associated with the severity of asthma (20). Further, steroid treatment appears to prevent or slow the development of fibrosis and the decline in lung function in asthma, but does not dramatically reverse existing changes (6). Our results suggest that early intervention with fluticasone and salmeterol could prevent or slow the remodeling response by their effect on HA production.


   Acknowledgments
 
This research was supported by a grant from GlaxoSmithKline.

Received in original form October 23, 2003

Received in final form January 29, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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