Tissue collection and cell culture.

Approval for all experimental protocols with human lung was provided by the Human Research Ethics Committees of The University of Sydney and the Sydney South West Area Health Service. HBECs were obtained from bronchial airways of volunteers with asthma or no lung disease and patients undergoing lung resection or transplantation. All donors provided written, informed consent.

Bronchial brushing through the flexible fiber-optic bronchoscope was used to collect epithelial cells from volunteers. The lung tissue obtained at thoracotomy was dissected, and the airways were isolated from macroscopically normal areas of lung. The epithelial layer was removed from the airways by macrodissection. After washing with HBSS, epithelial cells or tissue were placed in a tissue-culture flask in Ham's F-12 medium with growth supplements (9). The cells were maintained in Ham's F-12 and tested negative for mycoplasmal contamination. The experiments were performed in bronchial epithelial growth medium (BEGM; Cambrex Bio Science Walkersville, Walkersville, MD), as described previously (9). The experiments were performed with cells between passages 1 and 4. The cells from each individual were regarded as one primary cell culture. In this study, the primary cultures were categorized into two groups: the asthmatic and nonasthmatic group. The data from the asthmatic group were averaged, as were those from the nonasthmatic group. The number of primary cell cultures used within a group in each experiment is represented by n. The patient and volunteer demographics are shown in Table 1.

Cell experiments.

HBECs were seeded at 2 × 10⁴ cell/cm² in 48-well plates in triplicate for ECM ELISA and in 12-well plates for collection of RNA lysates. The cells were grown for 3 days in BEGM and then quiesced in bronchial epithelial basal medium (BEBM; Cambrex Bio Science) for 24 h. After quiescing, the medium was refreshed with BEGM in the absence or presence of TGF-β1 (0.1, 0.5, 1, and 5 ng/ml). The supernatants, ECM proteins, and total RNA lysates were collected from the HBECs at day 0 (quiesced in BEBM for 24 h), following 1, 2, or 3 days growth in BEGM. Samples for day 0 were collected at the time of stimulus addition after the cells had been quiesced for 24 h; the measurements from samples collected at this time point were considered as basal levels.

Specific pharmacological protein kinase inhibitors were used to explore the signaling pathways involved in TGF-β1-modulated ECM Fn deposition. After quiescing in BEBM for 24 h, the cells were pretreated with the MEK (MAPK/ERK) inhibitor PD98059 (10 μM), the JNK inhibitor SP600125 (10 μM), the PI3K inhibitor LY294002 (3 μM; Calbiochem, San Diego, CA), the p38 MAPK inhibitor SB239063 (3 μM), and the TGF-β type I receptor activin-like kinase 5 (ALK5) inhibitor SB431542 (1, 3, and 10 μM; Tocris, Ellisville, MO) in BEGM for 30 min before stimulation with and without TGF-β1 (1 ng/ml). We used these specific protein kinase inhibitors at concentrations that have been previously proven to be effective in human airway cells (9, 10, 18, 20, 41). ECM samples were collected after 3 days.

To test the effect of Fn on HBEC viability, proliferation, and the release of soluble cytokines and chemokines, human plasma Fn (BD Biosciences, Bedford, MA) was precoated on plates at 0, 1.58, 5, and 15.8 μg/ml in PBS, overnight at 37°C. The plates were washed with sterile PBS, and then the cells were seeded in BEGM. Samples were collected at days 1, 2, and 3.

The role of soluble Fn was also examined. Confluent and quiesced HBECs were treated with Fn at 0, 1.58, 5, and 15.8 μg/ml in BEGM. After 1, 2, and 3 days of incubation, cell viability and proliferation were determined, and supernatants were collected.

ECM Fn ELISA.

The ECM Fn ELISA was performed as described previously with some modifications (19). At the end of time points, the medium was removed from the plates. The plates were washed with PBS, and the cells were lysed with hypotonic ammonium hydroxide (0.016 M NH₄OH; Sigma, St. Louis, MO). At the time of analysis, human plasma Fn was used to generate a standard curve. A serial dilution of Fn at concentrations of 2,000, 1,000, 500, 250, 125, and 62.5 ng/ml was added to the empty wells of the plates, followed by incubation at 4°C overnight. The following day, the plates were washed with 0.05% Tween 20 in PBS and blocked with 1% BSA (Sigma) in PBS. An anti-human Fn antibody (clone 868A11; Millipore, Billerica, MA) and a polyclonal rabbit anti-mouse Ig/horseradish peroxidase (Dako, Glostrup, Denmark) were used. After final washing, a liquid substrate system, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma), was used to detect the amount of ECM Fn deposited by cells. The absorbance was measured immediately at 405 nm using a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). The readings from triplicate wells were averaged and the background absorbance subtracted. The data were expressed as nanograms per milliliter, which was calculated using the equation generated from the standard curve.

Soluble Fn ELISA.

The levels of Fn released by the cells into the supernatant were determined using a QuantiMatrix human Fn ELISA kit (Millipore), according to the manufacturer's instructions. The detection limit for soluble Fn was 3 ng/ml. A standard curve was constructed, the Fn concentration was interpolated from the standard curve, and GraphPad Prism (version 5.0) was used for further analysis.

Real-time PCR.

Real-time PCR was performed as described previously (9). The total RNA lysates were collected and extracted using a NucleoSpin RNA II kit, and Moloney murine leukemia virus RT was used for reverse transcription. A predeveloped, specific primer set for Fn, Hs00365058_m1, TaqMan Universal PCR Master Mix, and the StepOnePlus Real-Time PCR System was used for real-time PCR (Applied Biosystems, Branchburg, NJ). A predeveloped TaqMan reagent human 18S rRNA (Cat. #4319413; Applied Biosystems) was included in each real-time PCR reaction as an endogenous control. Data from the reactions were analyzed using StepOne Software, v 2.1 (Applied Biosystems).

ELISA for IL-6, VEGF, and PGE₂.

The IL-6, VEGF, and PGE₂ ELISAs were carried out following the manufacturers' instructions (IL-6, BD PharMingen, Franklin Lakes, NJ; VEGF, R&D Systems, Minneapolis, MN; and PGE₂, Cayman Chemical, Ann Arbor, MI).

Cell number, viability, and cytotoxicity assay.

At the end point of experiments, the cell number, viability, and cytotoxicity were determined using manual cell counting, a lactate dehydrogenase (LDH) assay, and/or a mitochondrial activity assay (MTT), respectively. The LDH assay was a means of measuring either the number of cells via total cytoplasmic LDH or membrane integrity (cytotoxicity) as a function of the amount of cytoplasmic LDH released into the medium. The MTT assay was a means of measuring the activity of living cells via mitochondrial dehydrogenases. Both assays were carried out according to the manufacturer's instructions (Sigma). The absorbance was measured using the SpectraMax M2 microplate reader. The data from each treatment were averaged and background absorbance subtracted, and GraphPad Prism (Version 5.0) was used for further analysis.

The cell-proliferation status and membrane integrity were also confirmed using CyQUANT direct cell proliferation assay, according to the manufacturer's instructions (Molecular Probes, Eugene, OR).

Statistical analysis.

Data were expressed as means ± SE for the number of HBEC cultures (n) stated and analyzed using GraphPad Prism (version 6.0). After testing for normal distribution and equal variance, the differences were assessed by unpaired Student's t-test, one-way or two-way ANOVA using Dunnett's or Sidak's multiple comparisons test, or Bonferroni post-tests with repeated measures as appropriate. P < 0.05 was considered statistically significant.

The expression of Fn from HBECs under basal conditions.

The levels of Fn mRNA expression were similar between asthmatic and nonasthmatic cells at days 0 and 1 (Fig. 1A). There was an increase in cell viability/proliferation measured by MTT assay and soluble Fn release from asthmatic HBECs at day 3, but no differences in soluble Fn release and cell number were seen when comparing the asthmatic and nonasthmatic cells at days 1, 2, and 3 (Fig. 1, B and D). However, asthmatic HBECs constitutively deposited greater amounts of ECM Fn than nonasthmatic cells (Fig. 1C).

TGF-β1 increased Fn production from HBECs.

Following stimulation with TGF-β1, the Fn mRNA expression was increased after 8 h, maximal at 24 h, and while beginning to decline from maximum, the levels were still above baseline at 48 and 72 h (n = 4; data not shown). Interestingly, after 24 h stimulation with TGF-β1, nonasthmatic HBECs expressed a greater amount of Fn mRNA compared with asthmatic cells (Fig. 2A). TGF-β1 increased both soluble and ECM Fn expression after 2 days of stimulation in nonasthmatic and asthmatic HBECs. Furthermore, the levels of ECM Fn produced by asthmatic cells were higher than those by nonasthmatic cells in the presence of TGF-β1 (Fig. 2, B and C, for day 3; days 1 and 2 data not shown), although the percentage of increase in ECM Fn induced by TGF-β1 was similar in nonasthmatic and asthmatic cells [at day 3, 5 ng/ml of TGF-β1; percentage increase over unstimulated 220.3 ± 54.6% for nonasthmatic, n = 7; 206.3 ± 44.0% for asthmatic, n = 5, respectively].

The HBEC viability and numbers modulated by TGF-β1 were monitored using MTT and CyQUANT direct cell proliferation assay. TGF-β1 reduced cell mitochondrial activity at days 1, 2, and 3 in nonasthmatic and asthmatic cells (Fig. 3A). It also decreased DNA-bound fluorescence intensity in both cell groups (Fig. 3B), which confirmed that the HBEC number was not increased by TGF-β1.

An ALK5 inhibitor SB431542 blocked TGF-β1-induced Fn expression.

The inhibitors for ERK (PD98058), PI3K (LY294002), JNK (SP600125), p38 MAPK (SB239063), and ALK5 (SB431542) were used to block individual signaling pathways, which may be involved in the TGF-β1-induced ECM Fn deposition in HBECs. PD98059, LY294002, SP600125, and SB239063 had no effect on ECM Fn deposition in the absence and presence of TGF-β1 in either nonasthmatic or asthmatic HBECs (Fig. 4, C–F). However, SB431542 inhibited TGF-β1-induced Fn mRNA expression in nonasthmatic and asthmatic HBECs (Fig. 4A). Furthermore, SB431542 blocked TGF-β1-stimulated ECM Fn deposition but had no influence on constitutive ECM Fn deposition in either nonasthmatic or asthmatic cells (Fig. 4B).

Fn increased HBEC proliferation and regulated proinflammatory mediator release.

To investigate the role of Fn in regulating cell proliferation and the release of proinflammatory mediators, the nonasthmatic HBECs were treated with Fn in two ways: 1) plasma Fn was precoated on the plates, and the cells were seeded on top of the Fn, and 2) the cells were grown to confluence and quiesced before plasma Fn was added as a stimulus.

When HBECs were seeded on the plate precoated with Fn, Fn increased cell viability at days 1, 2, and 3, as measured by MTT (Fig. 5A for day 2 in asthmatic and nonasthmatic cells; days 1 and 3 data not shown) and LDH (Table 2) assay. The precoated Fn induced HBEC proliferation (Fig. 5B), and this was also confirmed using a CyQUANT direct cell proliferation assay (Table 2). Furthermore, the rates of cell proliferation measured by MTT, LDH, cell counting, and CyQUANT assay were similar (Table 2). Precoated Fn had no effect on cytotoxicity of HBECs, as measured by released LDH (n = 12; data not shown).

Growth of the HBECs in tissue-culture wells precoated with Fn also altered the release of soluble factors important in asthma. Figure 6 illustrates IL-6 (A), PGE₂ (B), and VEGF (C) release in the presence or absence of precoated Fn after correction for cell number, as measured by MTT.

In contrast, soluble Fn, when added to confluent HBECs, had no effect on cell viability and proliferation, nor did it affect cytotoxicity (nonasthmatic n = 8 and asthmatic n = 3 for MTT and LDH assays; nonasthmatic n = 7 and asthmatic n = 4 for cell counting; data not shown). Similarly, soluble Fn did not alter the regulation of IL-6, PGE₂, and VEGF release in HBECs (nonasthmatic n = 9 and asthmatic n = 3 for IL-6 and VEGF release; nonasthmatic n = 5 and asthmatic n = 3 for PGE₂ release; data not shown).