Role of Abl in airway hyperresponsiveness and airway remodeling

Abl^-lox mice were a gift of Dr. Koleske of Yale University [16]. SM22^cre mice were purchased from The Jackson Laboratory. Abl^-lox mice (genetic background, 129/Svj) were crossed with SM22^cre mice on C57BL/6 background. These mice express Cre recombinase under control of a smooth muscle-specific SM22 promoter. As a consequence, this loxP flanked exon 5 of the abl gene was excised in smooth muscle cells (Figure 1A). The sequences of primers used to identify the genotype were: Primer 1, 5’-CTGTACGTGTCCTCCGAGAG-3’; Primer 2, 5’-CTTCAAGGTCTTCACGGCCA-3’; Primer 3, 5’-GATGTCTCTACAGGGTTAAGATTAAGAGCA-3’; Primer 4, 5’-TGTGCATAGCAGGAAGTCCTCCAGAG-3’; Primer 5, 5’-AGTTAACACACCTCCAGAGTGAGTGCCCT-3’.

Age- and sex-matched Abl^-lox (control) mice and Abl^sm−/− (smooth muscle specific knockout of abl gene) mice (6–7 weeks old) were sensitized by intraperitoneal injection of sterile LPS-free ovalbumin (OVA) (Pierce) or phosphate buffered saline (PBS, control) for three weeks, and challenged with intranasal instillations of OVA or PBS twice a week for eight weeks using pre-viously-described protocols [17] with minor modification (Figure 2). To measure airway resistance, mice were anesthetized with an intraperitoneal injection of pentobarbital sodium, tracheotomized, and connected to the FlexiVent system (SCIREQ, Montreal, Canada) on Day 77. Mice were mechanically ventilated at 150 breaths/minute with a tidal volume of 10 ml/kg and a positive end-expiratory pressure (PEEP) of 3.35 cm H₂O. Following baseline measurements, mice were challenged with methacholine (MCh) aerosol for 10 seconds at different doses. Airway resistance was measured for each mouse after inhalation of the aerosol. Dose–response curve was then determined. This study was approved by the Institutional Committee of Animal Care and Usage of Albany Medical College.

To assess the effects of inhibitors, BALB/c mice (6–7 weeks old) were purchased from The Jackson Laboratory. They were sensitized and challenged by OVA as described above. In addition, animals were intranasally instilled with 10 mg/kg GNF-5 or imatinib, or PBS 1 h before OVA instillation and 5 h after OVA instillation for last three weeks (Figure 2). Airway resistance in these mice was then assessed on Day 77.

Mice were euthanized by injection of pentobarbital (140 mg/kg). A segment of tracheas (4–5 mm in length) was immediately removed and placed in physiological saline solution (PSS) containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl₂, 0.8 mM MgSO₄, 25.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 5.6 mM glucose. The solution was aerated with 95%O₂-5%CO₂ to maintain a pH of 7.4. Two stainless steel wires were passed through the lumen of tracheal rings. One of the wires was connected to the bottom of organ baths and the other was attached to a Grass force transducer that had been connected to a computer with A/D converter (Grass). Tracheal segments were then placed in PSS at 37°C. Passive tension with 0.5 g was applied to each segment for 60 min. Contractile force in response to various treatments was then measured.

Human airway smooth muscle (HASM) cells were prepared from human bronchi and adjacent tracheas obtained from the International Institute for Advanced Medicine [15]. Human tissues were non-transplantable and consented for research. This study was approved by the Albany Medical College Committee on Research Involving Human Subjects. Briefly, muscle tissues were incubated for 20 min with dissociation solution [130 mM NaCl, 5 mM KCl, 1.0 mM CaCl₂, 1.0 mM MgCl₂, 10 mM Hepes, 0.25 mM EDTA, 10 mM D-glucose, 10 mM taurine, pH 7, 4.5 mg collagenase (type I), 10 mg papain (type IV), 1 mg/ml BSA and 1 mM dithiothreitol]. All enzymes were purchased from Sigma-Aldrich. The tissues were then washed with Hepes-buffered saline solution (composition in mM: 10 Hepes, 130 NaCl, 5 KCl, 10 glucose, 1 CaCl₂, 1 MgCl₂, 0.25 EDTA, 10 taurine, pH 7). The cell suspension was mixed with Ham’s F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin). Cells were cultured at 37°C in the presence of 5% CO₂ in the same medium. The medium was changed every 3–4 days until cells reached confluence, and confluent cells were passaged with trypsin/EDTA solution [15, 18, 19]. Smooth muscle cells within passage 5 were used for the studies.

Cells were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris–HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked with bovine serum albumin or milk for 1 h and probed with use of primary antibody followed by horseradish peroxidase-conjugated secondary antibody (Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Fisher Scientific) using the LAS-4000 Fuji Image System. Abl antibody was purchased from BD Biosciences and Santa Cruz Biotechnology. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from Fitzgerald (Acton, MA). The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multigauge Software). The luminescent signals from all immunoblots were within the linear range.

Mouse lungs were placed in frozen tissue-embedding medium (Neg 52, Richard-Allen Scientific) and cryosectioned using Cryostats (Richard-Allen Scientific). Tissue sections were fixed for 15 min in 4% paraformaldehyde, and were then washed three times in PBS buffer followed by permeabilization with 0.2% Triton X-100 dissolved in PBS for 5 min. These tissues were incubated with α-smooth muscle actin antibody (Sigma) or proliferating cell nuclear antigen (PCNA) antibody (Thermo Scientific) followed by appropriate secondary antibody conjugated to Alexa-488 or Alex-543 (Molecular Probes/Life Technologies). The sections were also counterstained with 4',6-diamidino-2-phenylindole to visualize the nucleus. The samples were viewed and digitally captured using a Leica microscope system (MDI 6000). All immunohistochemical measurements were performed by using the NIH ImageJ software.

All statistical analysis was performed using Prism 4 software (GraphPad Software, San Diego, CA). Comparison among multiple groups was performed by one-way analysis of variance followed by Tukey’s multiple comparison test. Differences between two groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

Our previous studies demonstrate that Abl regulates vascular smooth muscle contraction [11‐13]. To determine the role of Abl in airway smooth muscle, we generated SM22^creAbl^-lox mice, a mouse model with smooth muscle cell–specific disruption of the abl gene (Figure 1A). Genotyping and immunoblot analysis verified knockout of Abl in airway smooth muscle (Figure 1B and C). Previous studies by others demonstrate that SM22 is expressed in airway smooth muscle tissues [20‐22], which suggests that SM22 promoter is functional in airway smooth muscle. Our results are consistent with these studies.

Contractile responses of mouse tracheal rings to ACh stimulation were compared between Abl^sm−/− (smooth muscle knockout of Abl) mice and Abl^-lox (control) mice. As shown in Figure 3A, contractile responses of mouse tracheal rings to ACh were lower in Abl^sm−/− mice than in Abl^-lox mice, which was dose-dependent.

We also evaluated acute effects of the Abl pharmacological inhibitors imatinib (Gleevec, STI-571) [11, 23] and GNF-5 [24] on airway smooth muscle contraction. Treatment of mouse tracheal rings with imatinib significantly attenuated force development induced by ACh (Figure 3B). Likewise, GNF-5 had inhibitory effects on contraction of tracheal segments with slightly stronger potency (Figure 3B). Furthermore, treatment with imatinib or GNF-5 induced relaxation of tracheal rings precontracted by ACh (Figure 3C).

We evaluated the expression of Abl in airway tissues of OVA-sensitized and challenged mice, a well-recognized animal model mimicking allergen-induced asthma in humans [17]. As shown in Figure 4A, the amount of Abl was elevated in airway tissues of OVA-treated mice compared to naïve animals. However, the levels of GAPDH were similar in OVA-treated mice and naïve mice. The ratio of Abl/GAPDH in airway tissues was higher in OVA-treated mice than in naïve mice (Figure 4B). To validate this finding in human asthma, we assessed Abl expression in HASM cells from normal subjects and patients with severe asthma. The level of Abl was higher in asthmatic cells than in normal cells. The ratios of Abl/GAPDH in asthmatic cells were significantly higher as compared to normal cells (Figure 4A and B).

We used a chronic asthma animal model [17] to determine whether Abl in smooth muscle is involved in AHR. Briefly, Abl^-lox and Abl^sm−/− mice were sensitized by OVA for three weeks and challenged by OVA for eight weeks (Figure 2). Airway resistance in response to methacholine (MCh) inhalation was measured using the FlexiVent system. OVA sensitization and challenge induced a higher response to MCh inhalation in Abl^-lox mice as compared to Abl^-lox mice treated with PBS. However, airway resistance induced by MCh was lower in Abl^sm−/− mice sensitized and challenged by OVA than in Abl^-lox mice treated with OVA. The airway resistance was also lower in naïve Abl^sm−/− mice than in naïve Abl^-lox mice (Figure 5A).

We also assessed the effects of Abl knockout on airway smooth muscle hyperreactivity in vitro. Contractile force in isolated tracheal rings from OVA-treated Abl^-lox mice was greater compared to naïve Abl^-lox mice. However, active force of isolated tracheal rings from OVA-treated Abl^sm−/− mice was reduced compared to OVA-treated Abl^-lox mice. Contractile response of tracheal rings from naïve Abl^sm−/− mice was also lower compared to naïve Abl^-lox mice (Figure 5B).

We also evaluated the effects of the Abl inhibitors imatinib and GNF-5 on AHR in asthmatic animals. The OVA sensitization and challenge increased airway resistance in BALB/c mice as compared to BALB/c mice treated with PBS (Figure 6A). In contrast, the OVA-induced increase in airway resistance was reduced in the animals treated with imatinib or GNF-5 (Figure 6A). In addition, treatment with imatinib or GNF-5 inhibited the ACh-induced contraction in isolated mouse tracheal rings of OVA-sensitized and challenged mice (Figure 6B).

We noticed that airway resistance in response to MCh inhalation was slightly higher in BALB/c mice than in Abl^-lox mice sensitized and challenged by OVA (Figure 5A and Figure 6A). This is not surprising because BALB/c mouse strain is known to have skewed Th2 response compared to other mouse strains [25, 26].

To determine the role of Abl in the remodeling of airway smooth muscle, we assessed whether conditional knockout of Abl in smooth muscle affects the allergen-induced airway smooth muscle mass by determining the area of α-smooth muscle actin (a smooth muscle marker) staining in the airways of Abl^-lox and Abl^sm−/− mice sensitized and challenged with OVA.

The area of α-smooth muscle actin staining in the airways of Abl^-lox mice treated with OVA was greater than that in Abl^-lox mice treated with PBS, as evidenced by immunofluorescent analysis. In contrast, the area of actin staining in the airways of Abl^sm−/− mice treated with OVA was reduced as compared to Abl^-lox mice treated with OVA (Figure 7A & B). These results suggest that conditional knockout of Abl is able to attenuate the allergen-induced increase in airway smooth muscle mass. In addition, the fluorescent intensity of α-smooth muscle actin staining was higher in Abl^-lox mice treated with OVA compared to naïve Abl^-lox mice, suggesting higher α-smooth muscle actin expression in the remodeled airway in asthmatic models [6]. Furthermore, we determined the effects of imatinib or GNF-5 on airway smooth muscle growth. Treatment with the inhibitors attenuated an increase in airway smooth muscle mass in BALB/c mice sensitized and challenged by OVA (Figure 7B).

We also evaluated whether the knockout of Abl influences allergen-induced airway smooth muscle cell proliferation. Proliferating cell nuclear antigen (PCNA) is a critical protein that is expressed by proliferating cells in S phase of the cell cycle. Thus, it has been widely used as a marker of cell proliferation in the airways [8, 27]. The fluorescent intensity of PCNA colocalized with α-smooth muscle actin was greater in the airways of Abl^-lox mice treated with OVA compared with Abl^-lox mice treated with PBS. However, the intensity of PCNA costained with actin in the airways of Abl^sm−/− mice treated with OVA was lower than that in the airways of Abl^-lox mice treated with OVA (Figure 7A and C). Moreover, treatment with the Abl inhibitors imatinib or GNF-5 diminished the fluorescent intensity of PCNA in BALB/c mice treated with OVA (Figure 7C). These results indicate that Abl has a role in the allergen-induced airway smooth muscle proliferation.

As a consequence of allergic sensitization and challenge, inflammatory cells enter into the lungs and cytokine/chemokine levels are increased in the bronchoalveolar space of asthmatic patients and animal models, which are characteristic features of allergic airway inflammation [7, 28, 29]. To determine whether the smooth muscle-specific depletion of Abl affects recruitment of inflammatory cells, we determined total and differential cell counts of BALF in lungs of naïve and OVA-treated Abl^-lox mice and Abl^sm−/− mice.

OVA sensitization and challenge increased the numbers of total and differential cells in the lungs of Abl^-lox mice. However, the allergen-induced increase in cell numbers in the lungs in Abl^sm−/− mice was similar to that in Abl^-lox mice (Figure 8A and B).

We also evaluated the effects of the Abl inhibitors imatinib and GNF-5 on cell counts of BALF from mice treated with PBS or OVA. OVA sensitization and challenge increased total and differential cell counts of BALF from BALB/c mice. Treatment with imatinib and GNF-5 reduced the OVA-induced increase in inflammatory cell numbers (Figure 8C and D).

To determine the role of Abl in smooth muscle in the production of cytokine and chemokine, we evaluated the level of IL-13 and CCL2 (representative cytokine and chemokine in asthma pathology) [5, 30, 31] in the BALF in lungs of naïve and OVA-treated Abl^-lox and Abl^sm−/− mice. OVA sensitization and challenge increased the level of IL-13 and CCL2 in the BALF of Abl^-lox mice. In addition, the allergen-induced increase in IL-13 and CCL2 in the lungs of Abl^sm−/− mice was similar to those in Abl^-lox mice (Figure 9A and B). However, treatment with imatinib and GNF-5 diminished the OVA-induced increase in IL-13 and CCL2 in the lungs of BALB/c mice (Figure 9C and D).