Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells

Xiangde Liu1, Tadashi Kohyama1, Hangjun Wang2, Yun Kui Zhu3, Fu-Qiang Wen1, Hui Jung Kim1, Debra J. Romberger1, and Stephen I. Rennard1

1 Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, Omaha, Nebraska 68198; 2 Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; 3 Department of Respiratory Diseases, Jincheng Hospital, Lanzhou, China 710032


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-beta or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) E2 release. Decreased PGE2 release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PGE2 release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma.

asthma; interleukin; prostaglandin E2; cyclooxygenase; T helper 2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ASTHMA IS A CHRONIC INFLAMMATORY disease characterized by reversible airflow limitation and the presence of chronic inflammation of the airways (4, 10). In addition, the airways in asthma undergo characteristic structural alterations. This remodeling not only may contribute to the development of increased airway reactivity but may also lead to fixed airflow limitation.

A characteristic feature of inflammation in asthma is the accumulation of activated T helper 2 (Th2) lymphocytes (11, 24). These lymphocytes release a characteristic set of cytokines that are believed to play a major role in the airway reactivity that characterizes asthma. Fibroblasts are also able to respond to selected Th2 cytokines, suggesting the possibility that mesenchymal cell participation in airways remodeling may also be, at least in part, controlled by these cytokines (5, 18, 25).

Among the features that characterize wound healing and the development of fibrotic scar is tissue contraction. This process is thought to be mediated by fibroblast contraction of extracellular matrices. Such a process, developing circumferentially around an airway, could contribute to progressive, fixed airflow limitation. The current study, therefore, was designed to evaluate the hypothesis that Th2 cytokines might contribute to airway remodeling by modulating fibroblast contraction of extracellular matrices. To evaluate this hypothesis, the well-established in vitro system utilizing fibroblasts cultured in floating three-dimensional collagen gels was utilized.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Native type I collagen [rat tail tendon collagen (RTTC)] was extracted from rat tail tendons by a previously published method (1, 15). Briefly, tendons were excised from rat tails, and the tendon sheath and other connective tissues were removed carefully. Repeated washing with Tris-buffered saline was followed by dehydration and sterilization with 50%, 75%, 95%, and pure ethanol. Type I collagen was then extracted in 6 mM hydrochloric acid at 4°C. The supernatant was harvested by centrifugation at 2,000 g for 1 h at 4°C. Collagen concentration was determined by weighing a lyophilized aliquot from each lot of collagen solution.

Commercially available reagents were obtained as follows: interleukins (IL-1beta , IL-4, IL-5, IL-10, and IL-13), tumor necrosis factor-alpha , anti-TGF-beta 1 antibody (clone: 9016.2), anti-TGF-beta 2 antibody (clone: 8607.211), anti-TGF-beta 3 antibody (clone: 20724.1), TGF-beta 1, TGF-beta 2, TGF-beta 3, and biotinylated anti-TGF-beta 1, -beta 2, or -beta 3 antibodies were from R&D system (Minneapolis, MN). Hydroxyproline, collagenase, chloramine T, n-propanol, p-dimethylaminobenzaldehyde, prostaglandin E2 (PGE2), 3,3',5,5'-tetramethyl benzidine (TMB) were purchased from Sigma (St. Louis, MO). The PGE2 enzyme immunoassay (EIA) kit and cyclooxygenase (COX)-1 and COX-2 electrophoresis standards were purchased from Cayman Chemical (Ann Arbor, MI). Anti-COX-1 polyclonal antibody, anti-COX-2 polyclonal antibody, and horseradish peroxidase (HRP)-conjugated anti-goat-IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blot detection reagents [enhanced chemiluminescene (ECL)] were purchased from Amersham Pharmacia Biotech (Alameda, CA). HRP-streptavidin was from Zymed (San Francisco, CA).

Cell culture. Human fetal lung fibroblasts (HFL-1, lung, diploid, human) and human bronchial fibroblasts (HBF) were purchased from the American Type Culture Collection (Rockville, MD). Human airway smooth muscle cells (HASMC) prepared as described (19a) were obtained from Dr. Myron Toews (University of Nebraska Medical Center, Omaha, NE). The cells were cultured in Dulbecco's modified Eagle medium (DMEM; GIBCO, Rockville, MD) supplemented with 10% fetal calf serum (FCS), 100 µg/ml penicillin, 250 µg/ml streptomycin, 1.25 µg/ml fungizone, and 2 mM L-glutamine. Cells were refed three times weekly in 100-mm tissue culture dishes (Becton Dickinson Labware, Lincoln Park, NJ), and confluent cells were passaged at a 1:4 ratio.

Collagen gel preparation and contraction assay. Gels were prepared using a previously described method (16) by mixing RTTC, distilled water, and 4× concentrated DMEM so that the final mixture resulted in a physiological ionic strength, 1× DMEM, and a pH of 7.40. Cells were trypsinized (trypsin-EDTA; 0.05% trypsin, 0.53 mM EDTA-4Na, GIBCO) and suspended in serum-free DMEM (SF-DMEM) at a density of 107 cells/ml. Cells were then mixed with the neutralized collagen solution so that the final cell density in the collagen solution was 4 × 105 cells/ml, and the final concentration of collagen was 0.75 mg/ml. Aliquots (0.5 ml/well) of the mixture of cells in collagen were cast into each well of 24-well tissue culture plates (FALCON). After gelation was completed, normally within 20 min at room temperature, the gels were gently released from the 24-well tissue culture plates and transferred into 60-mm tissue culture dishes (three gels in each dish), which contained 5 ml of freshly prepared SF-DMEM with or without cytokines. The gels were then incubated at 37°C in a 5% CO2 atmosphere for 3-5 days, and the area of each gel was measured with an Optomax V image analyzer (Optomax, Burlington, MA) daily. Data were expressed as the percentage of area compared with the original gel size.

Hydroxyproline measurement. Hydroxyproline amount was quantified with a modification of a previously published method (22). Briefly, gels were centrifuged at 6,000 g for 5 min to eliminate the water and then dissolved with collagenase (0.25 mg/ml, 50 µl/gel). Supernatant was separated from cell pellets by centrifuging at 2,000 g for 5 min. Twenty microliters of this solution were gently mixed with 30 µl of 3.3 N sodium hydroxide, followed by autoclaving at 120°C for 20 min. Chloramine T reagent (450 µl per sample) was then applied, and the samples oxidized at room temperature for 25 min. After this, samples were gently mixed with 500 µl of freshly prepared Ehrlich's reagent and developed at 65°C for 20 min. Absorbance at 540-nm wavelength was measured with BIO-RAD microplate Reader Benchmark and MPMIII version 1.57 software.

DNA assay. To estimate cell number in three-dimensional collagen gels, DNA was assayed fluorometrically with Hoechst dye 33258 (Sigma) by a modification of a previously published method (12). Collagen gels were dissolved with collagenase (0.25 mg/ml in SF-DMEM, 0.5 ml/gel) for 2 h at 37°C. Cell pellets and supernatants were separated by centrifugation at 500 g for 10 min and frozen at -80°C overnight. Cells were then thawed and resuspended in 1 ml of distilled water. After this, the samples were briefly sonicated, and the suspensions were mixed thoroughly with 2 ml of TNE buffer (3 M NaCl, 10 mM Tris, 1.5 mM EDTA, pH 7.4) containing 2 µg/ml of Hoechst 33258. Fluorescence intensities were measured with a fluorescence spectrophotometer (LS-5, Perkin-Elmer, Foster City, CA) with excitation at 356 nm and emission at 458 nm. Cell number was then calculated by comparison to a standard curve relating cell number to fluorescence.

Measurement of TGF-beta isoforms and fibronectin by ELISA. TGF-beta and fibronectin in the media in which gels were floated and the supernatant solutions after gel digestion (see DNA assay) were determined by ELISA. Quantification of fibronectin was performed by an ELISA that is specific for human fibronectin and that does not detect bovine fibronectin (23). Quantification of TGF-beta isoforms was performed as follows. Plates were coated with monoclonal anti-TGF-beta 1, -beta 2, or -beta 3 antibodies, respectively, at 4°C overnight. After being washed three times (5 min each), standards and samples were added and incubated at room temperature for 2 h. All samples were assayed both untreated and after TGF-beta activation by acidification and neutralization. To accomplish this, a 500-µl sample was mixed with 100 µl of 1 N HCL and, after 10 min at room temperature, neutralized with 100 µl of 1.2 N NaOH/0.5 M HEPES. After being washed, bound antigen was detected after adding biotinylated-anti-TGF-beta 1, -beta 2, or -beta 3 antibodies for 1 h at room temperature. HRP-streptavidin (1:20,000 dilution) was then added for 1 h. Bound HRP was then detected with TMB. The reaction was stopped with 1 M H2SO4, and the product was quantified at 450 nm with a microplate reader.

Measurement of PGE2 by EIA. PGE2 concentration in the media in which gels were floated, as well as in the solubilized gels, was quantified using an EIA following the manufacturer's instructions (Cayman Chemical).

Western blotting. HFL-1 cells were mixed with neutralized type I collagen solutions and then cast into six-well tissue culture plates at a density of 4 × 105/ml (2 ml/well). After gelation, gels were fed with SF-DMEM with or without 25 ng/ml (2 ml/well) of IL-4, IL-5, IL-10, or IL-13. The gels were then released and allowed to contract in the medium for 48 h. Cell pellets were obtained by digesting the gels with collagenase (0.25 mg/ml in SF-DMEM) for 2 h at 37°C followed by centrifugation at 500 g for 10 min. Cell pellets were then treated with lysis buffer (35 mM Tris · HCl, pH 7.4, 0.4 mM EGTA, 10 mM MgCl2, 100 µg/ml aprotinin, 1 µM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 0.1% Triton X-100). Lysates were briefly sonicated on ice and centrifuged at 10,000 g for 3 min. The protein concentration in the supernatants was measured (BIO-RAD Protein Assay reagents, cat. no. 165-5035, Hercules, CA). Ten percent polyacrylamide gels were prepared, and SDS-polyacrylamide gel electrophoresis was performed under reducing conditions. Cell lysate proteins were diluted with 2× concentrated sample buffer (250 mM Tris · HCl, ph 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, 2% beta -mercaptoethanol) and heated at 95°C for 5 min before loading (5 µg/lane). The resolved proteins were transferred onto Immuno-Blot polyvinylidene difluoride (PVDF, BIO-RAD) following the manufacturer's instructions. The PVDF membrane was blocked and then incubated with 1 µg/ml of anti-COX-1 or anti-COX-2 polyclonal antibody (Santa Cruz Biotech). HRP-conjugated anti-goat-IgG was then allowed to bind, and, after being washed, the blots were probed with the ECL Western blot detection system according to the manufacturer's instructions (Amersham Pharmacia Biotech).

RNA isolation and complementary DNA synthesis. After 48 h of contraction, collagen gels were digested with collagenase (0.25 mg/ml in RNase-free PBS) at 37°C and 5% CO2 for 2 h. Total RNA was then extracted from the cell pellets with acid guanidine monothiocyanate, precipitated with isopropyl alcohol, and dissolved in TE buffer (10 mM Tris · HCl, pH 7.4, + 1 mM EDTA). The total RNA amount was quantified spectrophotometrically. To remove possible contaminating genomic DNA, 1 µg of total RNA was treated with DNase I following the manufacturer's instruction (GIBCO) for 15 min at room temperature, after which the reaction was stopped with 25 mM EDTA, and the sample was heated to 65°C for 10 min followed by 95°C for 5 min. For cDNA synthesis, ~400 ng of total RNA was transcribed with cDNA transcription reagents (Perkin-Elmer) with the use of random hexamers, and the cDNA was used for quantitative real-time PCR.

Quantitative real-time PCR. Gene expression was measured with the use of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer) as described previously (2). Primers and TaqMan probes were designed using the Primer ExpressTM 1.0 (Perkin-Elmer) software to amplify <150 base pairs. Probes were labeled at the 5' end with the reporter dye molecule 6-carboxy-fluorescein [FAM; emission (maximum absorbance) lambda max = 518 nm] and at the 3' end with the quencher dye molecule 6-carboxytetramethyl-rhodamine (TAMRA; emission lambda max = 582 nm). Target genes and the housekeeping gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] were simultaneously tested in duplicate or triplicate. Data were normalized to the amount of GAPDH and expressed as units per 105 GAPDH units. Real-time PCRs of cDNA specimens were conducted in a total volume of 50 µl with 1× TaqMan Master Mix (Perkin-Elmer), and primers at 300 nM and probes at 200 nM. Sequences used were as follows: COX-1 (forward), 5'-CAA TCA GAC AAG TGT TTT GGA AAG A-3'; COX-1 (reverse), 5'-TCC TCC GTT CTG CCA GCT T-3'; COX-1 (probe), 6FAM-TGC TCT GCC CTG TCA TCC ACC CTT-TAMRA. COX-2 (forward), 5'-GCT CAA ACA TGA TGT TTG CAT TCT-3'; COX-2 (reverse), 5'-GCT GGC CCT CGC TTA TGA-3'; COX-2 (probe), 6FAM-TGC CCA GCA CTT CAC GCA TCA GTT-TAMRA; GAPDH (forward), 5'-CCA GGA AAT GAG CTT GAG AAA GT-3'; GAPDH (reverse), 5'-CCC ACT CCT CCA CCT TTG AC-3'; GAPDH (probe), 6FAM-CGT TGA GGG CAA TGC CAG CCC-TAMRA. Thermal cycler parameters included 2 min at 50°C, 10 min at 95°C, and 40 cycles involving denaturation at 95°C for 15 s and annealing/extension at 60° for 1 min.

Statistical analysis. Each condition in the experiments of collagen gel contraction included three replicate gels, and the data from individual experiments are presented as the means ± SE of triplicates. Replica experiments, each with triplicate replicates, were performed on separate occasions. Student's t-test was performed to compare two group data. For multiple comparisons, ANOVA was performed followed by Tukey's test to make pair-wise comparisons. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Th2 cytokines on collagen gel contraction mediated by human lung mesenchymal cells. To investigate Th2 cytokine modulation of collagen gel contraction, the effect of IL-4, IL-5, IL-10, and IL-13 on collagen gel contraction mediated by HFL-1 cells was first tested by adding 10 ng/ml of each cytokine into the medium in which gels were floated. Both IL-4 and IL-13 significantly augmented the collagen gel contraction mediated by HFL-1 cells (Fig. 1), whereas IL-5 and IL-10 had no effect on the contraction. The augmenting effect of IL-4 and IL-13 was observed within 24 h after release, and it was due neither to degradation of the collagen (Fig. 2A) nor to increasing cell numbers in the gels (Fig. 2B). Furthermore, both IL-4 and IL-13 augmented the collagen gel contraction in a concentration-dependent manner over a range of 10-9-10-6 M (~0.1-10 ng/ml), although IL-4 was slightly more potent than IL-13 (Fig. 3). Similar effects of IL-4, IL-5, IL-10, and IL-13 were also seen for the gel contraction caused by adult HBF and adult HASMC, although IL-13 effect is less potent than IL-4 in HBF (Table 1).


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Fig. 1.   Effect of T helper 2 (Th2) cytokines on collagen gel contraction by human fetal lung (HFL)-1 cells. Native type I collagen gels were prepared as described in MATERIALS AND METHODS. After 20 min of polymerization, gels were released into 60-mm tissue culture dishes containing 5 ml serum-free DMEM (SF-DMEM) supplemented with or without 10 ng/ml of interleukin (IL)-4, IL-5, IL-10, and IL-13. Gel size was measured daily with an image analyzer. y-Axis, gel size expressed as percentage of initial gel size. Data were average of 3 separate experiments with triplicate gels in each. *P < 0.05 by t-test, comparing to control at respective time point.



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Fig. 2.   Effect of Th2 cytokines on collagen content and cell number in three-dimensional collagen gels. Native type I collagen gels were prepared and allowed to contract for 4 days in the presence or absence of varying cytokines as shown in Fig. 1. On day 4, hydroxyproline and DNA content in the gels were quantified as described in MATERIALS AND METHODS. A: x-axis, cytokine addition in the medium. B: y-axis, cell number converted from DNA (105 cells/gel); x-axis: cytokine addition in the medium.



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Fig. 3.   Concentration-dependent effect of IL-4 or IL-13 on collagen gel contraction by HFL-1 cells. Native type I collagen gels were prepared as described in MATERIALS AND METHODS. Gels were released into 60-mm tissue culture dishes containing 5 ml of SF-DMEM supplemented with varying concentrations of IL-4 or IL-13 as indicated in figure legends. Gels were allowed to contract at 37°C and 5% CO2 for 2 days. Gel size was measured with an image analyzer. y-Axis, gel size expressed as %initial size; x-axis, cytokine concentration (log M). *P < 0.05 by Tukey's method.


                              
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Table 1.   Differential effect of Th2 cytokines on collagen gel contraction by 3 lines of human airway cells

Cytokine effect on TGF-beta and fibronectin production by HFL-1 cells in three-dimensional collagen gels. It is known that TGF-beta and fibronectin are able to enhance collagen gel contraction by human lung fibroblasts. To determine whether TGF-beta and fibronectin mediate the augmenting effect of IL-4 and IL-13 on collagen gel contraction, TGF-beta 1, -beta 2, -beta 3, and fibronectin production in the contracting gel cultures was evaluated in response to IL-4 and IL-13. As shown in Table 2, fibronectin production by HFL-1 cells was not statistically different in response to IL-4 or IL-13. Similarly, there was no effect of the cytokines on TGF-beta 1 production. TGF-beta 2 and TGF-beta 3 were not detectable in any condition.

                              
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Table 2.   Effect of Th2 cytokines on TGF-beta 1 and fibronectin production by: HFL-1 cells in three-dimensional collagen gel culture

Cytokine effect on PGE2 production by HFL-1 cells in three-dimensional collagen gels. Because PGE2 is an inhibitor of collagen gel contraction by fibroblasts, we examined the effect of Th2 cytokines on PGE2 production in the three-dimensional culture system. The concentration of PGE2 was 30- to 40-fold higher in the collagen gels than in the surrounding culture medium. IL-4 significantly reduced the concentration in both the medium (Fig. 4A) and in the gels (Fig. 4B), whereas IL-13 reduced PGE2 concentration in the gels (Fig. 4B). IL-5 significantly increased the PGE2 production, and IL-10 had no effect (Fig. 4, A and B). Similar effects of these Th2 cytokines on PGE2 release were observed in HBF and HASMC (data not shown). Consistently, adding 10-7M (35.3 ng/ml) PGE2 into the medium in which gels were floated approximately restored the PGE2 before inhibition inhibited by IL-4 or IL-13 and blocked the augmented contraction induced by IL-4 or IL-13 (Fig. 5).


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Fig. 4.   Effect of Th2 cytokines on prostaglandin E2 (PGE2) production by HFL-1 cells. Native type I collagen gels were prepared and released into 60-mm tissue culture plate containing 5 ml SF-DMEM with or without cytokines. After 48 h of contraction, the media surrounding the gels were harvested, and gels were dissolved with collagenase (0.25 mg/ml, 500 µl/gel). Total PGE2 was then quantified in the medium and solubilized gels by enzyme immunoassay (EIA). A: PGE2 concentration in the surrounding medium. B: PGE2 concentration in the solubilized gels. x-Axis, cytokines in the medium. *P < 0.05 by t-test, comparing to control.



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Fig. 5.   Effect of exogenous PGE2 on IL-4- or IL-13-augmented gel contraction. Native type I collagen gels were prepared and released into 60-mm dishes containing SF-DMEM with varying concentration of PGE2 in the presence or absence of IL-4 or IL-13. Gel size was measured with an image analyzer on day 3. y-Axis, gel size expressed as %initial size; x-axis, PGE2 concentration (log M).

Indomethacin also significantly augmented HFL-1 gel contraction (Fig. 6) and inhibited PGE2 production (Fig. 7). Both IL-4 and IL-13, however, further enhanced the indomethacin-induced augmentation of gel contraction (Fig. 6). In contrast, neither cytokine had an effect on the near complete inhibition of PGE production induced by indomethacin.


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Fig. 6.   Effect of indomethacin, IL-4, and IL-13 on collagen gel contraction by HFL-1 cells. Native type I collagen gels were prepared and released into 60-mm dishes containing SF-DMEM with or without 2 µM indomethacin in the presence or absence of IL-4 or IL-13 (10 ng/ml). Gel size was measured with an image analyzer on day 3. y-Axis, gel size expressed as %initial size (mean ± SE); x-axis, addition of cyclooxygenase (COX) inhibitor; open bars: SF-DMEM; hatched bars, +IL-4 (10 ng/ml); solid bars, +IL-13 (10 ng/ml). *P < 0.05. **P < 0.01 by t-test.



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Fig. 7.   Effect of indomethacin, IL-4 and IL-13 on PGE2 production by HFL-1 cells. Native type I collagen gels were prepared and released into 60-mm dishes containing SF-DMEM with or without 2 µM indomethacin in the presence or absence of IL-4 or IL-13 (10 ng/ml). Media in which gels were floated were harvested after 3 days, and PGE2 amounts were measured by EIA as described in MATERIALS AND METHODS. y-Axis, PGE2 concentration expressed as nM (mean ± SE); x-axis, addition of COX inhibitor.

Cytokine regulation on COX-1, COX-2 protein production. Cyclooxygenases are responsible for the synthesis of prostaglandins. Because IL-4 and IL-13 significantly inhibited PGE2 production in the collagen gel culture system, the role of IL-4 and IL-13 in modulating cyclooxygenase expression was further investigated. HFL-1 cells were cultured in the three-dimensional collagen gels and allowed to contract for 48 h in the presence or absence of IL-4, IL-5, IL-10, or IL-13. Cell lysate protein was then immunoblotted for COX-1 and COX-2. As shown in Fig. 8, IL-4 and IL-13 inhibited COX-1 and COX-2 expression, whereas IL-5 increased enzyme expression (Fig. 8).


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Fig. 8.   Effect of Th2 cytokines on COX-1 (A) and COX-2 (B) protein content in HFL-1 in three-dimensional collagen gel culture. HFL-1 cells were cast into collagen gels and allowed to contract in SF-DMEM in the presence or absence of IL-4, IL-5, IL-10, or IL-13. After 48 h, gels were digested with collagenase, protein was extracted from the cell pellets, and immunoblots with antibody specific for COX-1 or COX-2 were performed. Electrophoresis standard of COX-1 and COX-2 was used as positive controls. The culture conditions for each lane are indicated in the figure.

Cytokine regulation of COX-1 and COX-2 mRNA. To further evaluate the mechanism of cytokine regulation of cyclooxygenase expression, the effect of Th2 cytokines on COX-1 and COX-2 gene expression was determined by quantitative real-time PCR. Consistent with the effect on protein expression assessed by immunoblot, IL-4 and IL-13 inhibited COX-1 and COX-2 mRNA expression (Fig. 9). In contrast, IL-5 stimulated the expression of COX-1 and COX-2 mRNA (Fig. 9).


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Fig. 9.   Effect of Th2 cytokines on COX-1 and COX-2 mRNA levels by HFL-1 cells in three-dimensional collagen gel culture. HFL-1 cells were cast into collagen gels and allowed to contract in SF-DMEM in the presence or absence of IL-4, IL-5, IL-10, or IL-13. After 48 h of contraction, gels were digested with collagenase, and total RNA was extracted from the cell pellets and used to quantify mRNA by real time quantitative PCR. A: COX-1 mRNA. B: COX-2 mRNA. y-Axis, mRNA amount that normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed per 105 GAPDH units. *P < 0.05 by t-test, comparing to control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cytokines IL-4, IL-5, IL-10, and IL-13, sometimes termed Th2 cytokines, play an important role in the development of chronic airway inflammation in asthma (11, 24, 32). In the current study, we report that IL-4 and IL-13 may also modulate mesenchymal cell-mediated tissue remodeling. Both IL-4 and IL-13 augmented contraction of native type I collagen gels populated by HFL-1 fibroblasts, normal HBF, and HASMC. In contrast, IL-5 and IL-10 did not have such an effect on any cell type tested. Much of the augmenting effect of IL-4 or IL-13 on collagen gel contraction by human lung fibroblasts can be attributed to decreasing endogenous PGE2 release through reducing COX-1 and COX-2 levels. In contrast, production of TGF-beta 1, -beta 2, -beta 3 and fibronectin was not affected by these cytokines.

IL-4 is a pleiotropic cytokine produced by a subset of CD4+ T cells, basophils, eosinophils, and mast cells. IL-13 is also produced by a variety of cell types including Th2 cells, basophils, and mast cells (13, 26, 30). Both cytokines have been demonstrated to drive Th2 type immune responses. In this context, the two cytokines show a high degree of homology in their amino acid sequence, and their receptors share a common subunit. The current study suggests that these two cytokines may also share the ability to modulate tissue remodeling through augmenting mesenchymal cell mediated contraction of extracellular matrix.

Previous studies also support the concept that IL-4 and IL-13 can participate in tissue remodeling (14, 34). Human lung fibroblasts express receptors for IL-4 and IL-13 (6). IL-4 has been reported to stimulate fibroblast chemotaxis (21). In addition, IL-4 can stimulate fibroblast proliferation (34). Finally, IL-4 can stimulate fibroblast production of extracellular matrix components including collagen, fibronectin, and tenascin (3, 14, 20, 29). Similarly, IL-13 has been demonstrated to upregulate total collagen production as well as procollagen Ialpha 1 and IIIalpha 1 gene expression in normal and keloid skin fibroblasts (19). The current study extends these observations and demonstrates that extracellular matrix restructuring by human airway mesenchymal cells can also be driven by both IL-4 and IL-13. Both cytokines comparably augmented collagen gel contraction by HFL-1 and HASMC cells. In HBF collagen gel contraction, however, IL-13 is less potent than IL-4. The variance of cell types and receptor expression may account for the differences.

Both normal tissue repair and the development of fibrosis are characterized by contraction of extracellular matrices. This contraction may serve several functional roles. It may serve to restrict scar size and to increase tensile strength. This may have significant benefits in wound repair. Excessive contraction of parenchymal tissues, which characterizes fibrosis, however, can lead to architectural derangements and can compromise tissue function. Contraction may, in addition, be one means for the resolution of granulation tissue and, therefore, may help limit the accumulation of excess connective tissue. In this context, several recent studies have suggested that contraction may lead to the induction of apoptosis (8, 33). Therefore, the ability of IL-4 and IL-13 to augment contraction of extracellular matrices may play a role both in the resolution of provisional matrix deposited after injury and in tissue derangements, which occur in fibrotic processes.

IL-4 and IL-5 have been suggested to play an important role in the development of asthma by modeling airway hyperreactivity and inflammation. Previous studies have indicated that decreased PGE2 in the airway coincided with airway hyperreactivity (7). Similarly, aspirin-induced asthma is associated with increased cystinyl leukotrienes as well as decreased PGE2 release (28). In the current study, IL-5 alone increased cyclooxygenases and PGE2 release but did not affect collagen gel contraction. In the presence of IL-4, however, IL-5-induced PGE2 increase was blocked, and the effect on collagen gel contraction was enhanced (data not shown). IL-4 inhibition of PGE2 production by human lung fibroblasts may contribute to asthma by modulating both airway reactivity and remodeling.

A number of other mediators have been described that can modulate mesenchymal cell mediated matrix contraction. Among these are TGF-beta and platelet-derived growth factor (PDGF), two mediators that are thought to play prominent roles in tissue repair (9, 17). Both of these mediators are potent augmenters of tissue contraction. The current study suggests that IL-4 and IL-13 can exert effects, at least with regard to collagen gel contraction, that are similar to TGF-beta and PDGF.

Previous studies have demonstrated that fibroblasts cultured in type I collagen gels produce PGE2, which can function as an endogenous inhibitor of collagen gel contraction (27). In the current study, IL-4 and IL-13 appeared to augment collagen gel contraction in large part by the inhibition of PGE2, which functions as a paracrine inhibitor. The mechanism by which IL-4 and IL-13 inhibit PGE2 appears to be due to inhibition of expression of the cyclooxygenase enzymes, which are the key regulatory steps in conversion of arachidonic acid to prostaglandins. In this context, IL-4 and IL-13 differ significantly from TGF-beta .

In contrast to the inhibitory effect on cyclooxygenase expression and PGE2 production induced by IL-4 and IL-13, TGF-beta stimulates PGE2 production by increasing cyclooxygenase expression (31). This suggests that TGF-beta stimulates contraction while, at the same time, stimulating an endogenous downregulator of the contractile process. This raises the interesting possibility that a repair response mediated by TGF-beta might be significantly altered in the concurrent presence of IL-4 or IL-13, which might disrupt endogenous paracrine counterregulatory pathways. Limited studies have been performed with the concurrent presence of IL-4 and TGF-beta . Interestingly, collagen gel contraction is greater in the presence of both mediators than that observed with either mediator alone. The mechanisms of the interaction are currently under investigation.

In the current study, IL-4 and IL-13 were able to augment collagen gel contraction even in the presence of indomethacin, which completely blocked PGE production. This suggests that although much of the effect of IL-4 and IL-13 is likely due to modulating PGE2 autoregulation, additional effects leading to augmented contraction are also, in part, responsible for the augmented contraction. The mechanism for these additional effects remains to be defined.

In summary, the current study demonstrates that IL-4 and IL-13 can modulate tissue repair by augmenting mesenchymal cell mediated contraction of extracellular matrices. Through such mechanisms, IL-4 and IL-13 may contribute to the repair processes that follow injury and may contribute to the development of pathological lesions such as fibrosis. By altering the endogenous paracrine pathways that regulate mesenchymal cell-mediated matrix contraction, IL-4 and IL-13 also have the possibility of disrupting normal regulatory pathways activated by other mediators of repair such as TGF-beta . Such interactions may play important roles in tissue alterations that develop in conditions such as asthma.


    ACKNOWLEDGEMENTS

We acknowledge Dr. Paula Belloni from Roche Bioscience for kindly providing us with GAPDH probes and primers. We also greatly appreciate and acknowledge the assistance in manuscript preparation by Lillian Richards and Mary Tourek.


    FOOTNOTES

This work was supported in part by National Institute of Heart, Lung, and Blood Grant RO1 HL-64088-01 and the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska.

Address for reprint requests and other correspondence: S. I. Rennard, Univ. of Nebraska Medical Center, 985125 Nebraska Medical Center, Omaha, NE 68198-5125 (E-mail: srennard{at}unmc.edu).

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

First published November 30, 2001;10.1152/ajplung.00321.2001

Received 13 August 2001; accepted in final form 27 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 282(5):L1049-L1056
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