Intranasal administration of recombinant progranulin inhibits bronchial smooth muscle hyperresponsiveness in mouse allergic asthma
Abstract
Progranulin (PGRN) is a growth factor with multiple biological functions and has been suggested as an endogenous inhibitor of Tumor necrosis factor-α (TNF-α)-mediated signaling. TNF-α is believed to be one of the important mediators of the pathogenesis of asthma, including airway hyperresponsiveness (AHR). In the present study, effects of recombinant PGRN on TNF-α-mediated signaling and antigen-induced hypercontractility were examined in bronchial smooth muscles (BSMs) both in vitro and in vivo. Cultured human BSM cells (hBSMCs) and male BALB/c mice were used. The mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also received intranasal administrations of recombinant PGRN into the airways 1 h before each antigen inhalation. In hBSMCs, PGRN inhibited both the degradation of IκB-α (an index of NF-κB activation) and the upregulation of RhoA (a contractile machinery-associated protein that contributes to the BSM hyperresponsiveness) induced by TNF-α, indicating that PGRN has an ability to inhibit TNF-α-mediated signaling also in the BSM cells. In BSMs of the repeatedly antigen-challenged mice, an augmented contractile responsiveness to acetylcholine with an upregulation of RhoA was observed: both the events were ameliorated by pretreatments with PGRN intranasally. Interestingly, a significant decrease in PGRN expression was found in the airways of the repeatedly antigen-challenged mice rather than those of control animals. In conclusion, exogenously applied PGRN into the airways ameliorated the antigen-induced BSM hyperresponsiveness, probably by blocking TNF-α-mediated response. Increasing PGRN levels might be a promising therapeutic for AHR in allergic asthma.
INTRODUCTION
Asthma is now estimated to affect around 235 million people worldwide (www.who.int/features/factfiles/asthma/en/). Enhanced airway responsiveness to nonspecific stimuli is a cardinal feature of bronchial asthma. The feature, called airway hyperresponsiveness (AHR), has come to be controlled mostly but not completely by the spread use of inhaled corticosteroids and long-acting β2-agonists. However, the underlying mechanism of AHR in asthmatics is not fully understood. This may be one of the reasons that the deaths due to asthma are still observed.
Tumor necrosis factor-α (TNF-α), one of the pro-inflammatory cytokines, is elevated in the airways of patients (3) and animal models (35, 49) of bronchial asthma. Evidence also suggests that TNF-α is directly linked to AHR (34, 42). Incubation of airway smooth muscle with TNF-α augmented its contraction induced by contractile agonists (1, 39). In addition, systemic treatment with antibodies against TNF-α inhibited AHR in a mouse model of allergic bronchial asthma (16). It is thus possible that TNF-α and its signaling molecules are therapeutic targets for asthma.
Progranulin (PGRN), also known as acrogranin, proepithelin, GP88, granulin/epithelin precursor, or PC cell-derived growth factor, is a growth factor with multiple biological functions (26) and is widely expressed in mammalian cells, including airway epithelial cells (17). Increasing evidence suggest that PGRN is a potent anti-inflammatory molecule (28, 43, 46, 50, 51, 54, 56). The anti-inflammatory activity of PGRN has also been suggested in acute lung inflammation induced by lipopolysaccharide (25, 53). The anti-inflammatory effects of PGRN might be mediated, at least in part, by blocking TNF-α binding to its receptors (43). It is thus possible that PGRN has the ability to ameliorate TNF-α-associated airway inflammatory disorders such as allergic asthma. Although the functional role of PGRN in asthma remains unknown to date, a downregulation of endogenous PGRN in the airways may also contribute to the pathogenesis of the disease.
Given the importance of TNF-α in the pathogenesis of AHR and the TNF-α blocking activity of PGRN, we hypothesized that PGRN might represent a novel treatment for allergic asthma. In the present study, effects of recombinant PGRN on TNF-α-mediated signaling and antigen-induced hypercontractility were examined in bronchial smooth muscles (BSMs) both in vitro and in vivo. Change in the expression level of PGRN was also determined in the airways of mice with allergic asthma.
METHODS
Cell culture and sample collection.
Normal human BSM cells (hBSMCs; a male donor, purchased from Cambrex Bio Science Walkersville, Walkersville, MD) were maintained in SmBM medium (Cambrex) supplemented with 5% fetal bovine serum, 0.5 ng/ml human epidermal growth factor (hEGF), 5 µg/ml insulin, 2 ng/ml human fibroblast growth factor-basic (hFGF-b), 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. Cells were maintained at 37°C in a humidified atmosphere (5% CO2), fed every 48–72 h, and passaged when cells reached 90–95% confluence. Then the hBSMCs (passages 5–7) were seeded in six-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at a density of 3,500 cells/cm2, and when 80–85% confluence was observed, cells were cultured without serum for 24 h before addition of recombinant human TNF-α (PeproTech, Rocky Hill, NJ) and/or PGRN (Adipogen International, San Diego, CA). At the indicated time after treatment, cells were washed with PBS, immediately collected and disrupted with ×1 SDS sample buffer (150 µl/well), and used for Western blot analyses. For RNA extraction, the lysis solution (350 µl/well) provided by Vantage Total RNA Purification Kit (Origene Technologies, Rockville, MD) was added directly to the washed cells.
Animals.
A total of 84 male BALB/c mice were purchased from Tokyo Laboratory Animals Science Co. (Tokyo, Japan) and housed in a pathogen-free facility. These mice were randomly divided into four groups as described below. All animal experiments were approved by the Animal Care Committee of the Hoshi University (Tokyo, Japan).
Sensitization and antigenic challenge.
Preparation of a murine model of allergic bronchial asthma, which has an in vivo AHR (30), was performed as described previously (8, 13–15). In brief, BALBc mice (8 wk of age) were actively sensitized by intraperitoneal injections of 8 µg ovalbumin (OA) (Seikagaku, Tokyo, Japan) with 2 mg Imject Alum (Pierce Biotechnology, Rockfold, IL) on day 0 and day 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/ml) for 30 min on days 12, 16, and 20. A control group of mice received the same immunization procedure, but inhaled saline aerosol instead of the OA challenge. The aerosol was generated with a compressor nebulizer (MiniElite, Philips Respironics) and introduced to a Plexiglas chamber box (130 × 200 mm, 100 mm height) in which the mice were placed. Twenty-four hours after the last OA challenge, mice were euthanized by exsanguination from the abdominal aorta under urethane (1.6 g/kg ip; Sigma-Aldrich, St. Louis, MO) anesthesia.
Intranasal administration of PGRN.
Recombinant mouse PGRN was intranasally administered 1 h before each antigen inhalation by the method previously described (5, 6, 8, 11). In brief, mice were anesthetized with sevoflurane (Maruishi Pharmaceutical, Osaka, Japan) and allowed to breathe spontaneously. Sterile PBS (20 µl; control) or recombinant mouse PGRN (100 ng in 20 µl PBS; AdipoGen) was intranasally instilled into the airways of each animal. Under these conditions, the reagent was successfully distributed in wide area of airways by the intranasal administration (5).
Analyses of bronchoalveolar lavage fluids.
After the exsanguinations, the chest of each animal was opened, and a 20-gauge blunt needle was tied into the proximal trachea. Bronchoalveolar lavage (BAL) fluid was obtained by intratracheal instillation of 1 ml/animal of PBS (pH 7.5, room temperature) into the lung while it was kept located within the thoracic cavity. The lavage was reinfused into the lung twice before final collection. BAL cells were isolated by centrifugation at 500 g. The resultant pellet was resuspended in 500 µl of 10% formaldehyde and incubated for 10 min. Then the cells were washed by PBS and resuspended in 500 µl of PBS. An aliquot of BAL cell suspension was used for cell counts with a hemocytometer. The resultant supernatants of the lavage fluids were subjected to TNF-α and PGRN analyses by using a mouse TNF-α ELISA kit (PeproTech) and a mouse progranulin ELISA kit (RayBiotech, Norcross, GA), respectively, according to the manufacturer’s instructions.
Determination of BSMs responsiveness.
Mice were killed by exsanguination from abdominal aorta under urethane (1.6 g/kg ip) anesthesia, and the airway tissues under the larynx to lungs were immediately removed. About a 3-mm length of the left main bronchus (~0.5-mm diameter) was isolated, and epithelium was removed by gently rubbing with sharp tweezers (12, 15). Removal of the epithelium was confirmed histologically in our preliminary study. The resultant tissue ring preparation was then suspended in a 5-ml organ bath by two stainless-steel wires (0.2-mm diameter) passed through the lumen. For all tissues, one end was fixed to the bottom of the organ bath while the other was connected to a force-displacement transducer (TB-612T, Nihon Kohden, Tokyo, Japan). Contractile forces were recorded isometrically by using the transducer that was connected to a bridge amplifier and to the PowerLab 4/26 data acquisition system (Model ML846: ADInstruments, New South Wales, Australia). A resting tension of 0.5 g was applied. The buffer solution contained modified Krebs-Henseleit solution with the following composition (in mM): NaCl 118.0, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 25.0, KH2PO4 1.2, and glucose 10.0. The buffer solution was maintained at 37°C and oxygenated with 95% O2-5% CO2. After the equilibration period, the concentration-response curve to acetylcholine (ACh: 10−7–10−3 M in final concentration) was constructed cumulatively. A higher concentration of ACh was successively added after attainment of a plateau response to the previous concentration.
Protein samples of mouse bronchial tissues.
Protein samples of BSM tissues were prepared as previously described (8, 13–15) with minor modification. In brief, both left and right main bronchi were removed and immediately soaked in ice-cold, oxygenated Krebs-Henseleit solution. The bronchi were carefully cleaned of adhering connective tissues and blood vessels under a stereomicroscopy. Then the bronchial tissue segments were homogenized in 200 µl of ×1 SDS sample buffer (Wako Pure Chemical Industries, Osaka, Japan) by using a disposable homogenizer BioMasher-II (Nippi, Tokyo, Japan). The tissue homogenate was then centrifuged (3,000 g, 4°C for 10 min), and the resultant supernatant was stored at −85°C until use.
RNA extraction from mouse bronchial tissues.
Both left and right main bronchi were isolated as described above. Then the bronchial tissue segments were homogenized in 350 µl of lysis solution provided by Vantage total RNA purification kit (Origene). Total RNAs including small RNAs were extracted using the kit according to the manufacturer’s instructions.
Western blot analyses.
Protein samples were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with EzBlock Chemi (Atto, Tokyo, Japan), the PVDF membrane was incubated with the primary antibody. The primary antibodies used in the present study were polyclonal rabbit anti-RhoA (1:2,500 dilution) and anti-IκB-α (1:500 dilution) antibodies. Then the membrane was incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:2,500 dilution), detected using EzWestBlue (Atto) and analyzed by a densitometry system. Detection of the house-keeping gene was also performed on the same membrane by using monoclonal mouse anti-GAPDH (1:10,000 dilution, for mouse samples) or polyclonal rabbit anti-GAPDH (1:10,000 dilution, for hBSMC samples) to confirm the same amount of proteins loaded. All of the antibodies used were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The specificity of each antibody used in the present study was validated previously (8, 13–15).
Determination of active form of RhoA in BSM.
The active form of RhoA, GTP-bound RhoA, in BSMs was measured by RhoA pull-down assay as described previously (15). In brief, the isolated main bronchial tissues were equilibrated in oxygenated Krebs-Henseleit solution at 37°C for 1 h. After the equilibration period, the tissues were stimulated with ACh (10−3 M for 10 min) and were quickly frozen with liquid nitrogen. The tissues were then lysed in lysis buffer with the following composition (mM): HEPES 25.0 (pH 7.5), NaCl 150, IGEPAL CA-630 1%, MgCl2 10.0, EDTA 1.0, glycerol 10%, NaF 25.0, sodium orthovanadate 1.0, and peptidase inhibitors. Active RhoA in tissue lysates (200 µg protein) was precipitated with 25 µg GST-tagged Rho binding domain (amino acids residues 7–89 of mouse rhotekin; Upstate, Lake Placid, NY), which was expressed in Escherichia coli and bound to glutathione-agarose beads. The precipitates were washed three times in lysis buffer, and after adding the SDS loading buffer and boiling for 5 min, the bound proteins were resolved in 15% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-RhoA antibody as described above.
Quantitative RT-PCR analyses.
Expression levels of mRNAs were determined by quantitative RT-PCR analysis. Reverse transcription reactions were performed using a cDNA synthesis kit (HP100042: Origene) according to the manufacturer’s instructions. The cDNA products from each sample were then subjected to RT-PCR analyses by using StepOne RT-PCR system (Applied Biosystems, Foster City, CA) with Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The reactions were incubated in a 48-well optical plate at 95°C for 20 s, following by 43 cycles of 95°C for 3 s, and 60°C for 30 s. The PCR primer sets used are shown in Table 1, which were designed from published sequences.
| Gene Name | Accession No. | Sequence | Amplicon Size | |
|---|---|---|---|---|
| Mouse PGRN | NM_008175 | sense | 5′-AGTGCAGATGGGAAATCCTG-3′ | |
| antisense | 5′-CAACCATAATGCAGCAGGTG-3′ | 102 bp | ||
| Mouse CCL2 | NM_008084 | sense | 5′-CCACTCACCTGCTGCTACTC-3′ | |
| antisense | 5′-AGCTTGGTGACAAAAACTACAGC-3′ | 119 bp | ||
| Mouse GAPDH | NM_008084 | sense | 5′-CCTCGTCCCGTAGACAAAATG-3′ | |
| antisense | 5′-TCTCCACTTTGCCACTGCAA-3′ | 100 bp | ||
Data and statistical analyses.
In the RT-PCR analyses, the comparative threshold cycle (CT) method was used for relative quantification of the mRNA for PGRN and Ccl2 (13). Differences in the CT values (ΔCT) between the target gene and GAPDH were calculated to determine the relative expression levels by using the following formula: ΔΔCT = (ΔCT of the treated sample) – (ΔCT of the control sample). The relative expression level between the samples was calculated according to the equation 2-ΔΔCT.
All the data are expressed as means ± SE. Statistical significance of difference was determined by unpaired Student’s t-test, one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple comparison, or two-way ANOVA (Prism 5 for Mac OS X; GraphPad Software, La Jolla, CA). A value of P < 0.05 was considered significant.
RESULTS
Inhibition of the TNF-α-induced IκB-α degradation by PGRN in cultured hBSMCs.
Reportedly, PGRN is known to act on receptors for TNF-α and inhibit TNF-α-mediated intracellular signaling (43). To confirm that PGRN also inhibits TNF-α-mediated signaling in hBSMCs, cells were treated with TNF-α in the absence or presence of PGRN. Degradation of IκB-α was used as an index of TNF-α-mediated NF-κB activation (52). As shown in Fig. 1A, incubation of hBSMCs with TNF-α caused a significant decrease in IκB-α protein level. The TNF-α-mediated decrease in IκB-α protein was attenuated by coincubation with PGRN (Fig. 1B), indicating that PGRN has an ability to inhibit TNF-α-mediated activation of NF-κB in hBSMCs. PGRN only had no effect on IκB-α protein abundance (of Fig. 1, A and B, open columns).
Fig. 1.Effects of recombinant progranulin (PGRN) on tumor necrosis factor-α (TNF-α)-induced downregulation of inhibitor of κB-α (IκB-α: A and B) and upregulation of RhoA (C and D) in cultured human bronchial smooth muscle cells. After a starvation period, the cells were incubated with TNF-α (0, 10, or 30 ng/ml) for 24 h in the absence (A and C) or presence of PGRN (100 ng/ml: B and D), and the expression levels of IκB-α and RhoA were determined by immunoblottings. Representative blots for IκB-α, RhoA, and GAPDH are shown in respective panels at top. The bands were analyzed by a densitometer, and the data are summarized in the panels at bottom. Results are presented as means ± SE from five to six independent experiments. *P < 0.05 vs. control (0 ng/ml TNF-α) by one-way ANOVA with post hoc Dunnett.
Inhibition of the TNF-α-induced upregulation of RhoA by PGRN in cultured hBSMCs.
Consistent with our previous studies (23, 24), treatment of hBSMCs with TNF-α caused an increase in RhoA protein in a concentration-dependent manner (Fig. 1C). A significant increase in RhoA protein was observed when cells were treated with 30 ng/ml of TNF-α (Fig. 1C). The upregulation of RhoA induced by TNF-α is reportedly mediated via an activation of NF-κB in hBSMCs (23, 24). To elucidate the functional inhibition by PGRN, its effect on the TNF-α-induced upregulation of RhoA was determined. As shown in Fig. 1D, the RhoA upregulation induced by TNF-α was inhibited by coincubation with human recombinant PGRN. The RhoA upregulation induced by TNF-α was also observed when cells were pretreated with PGRN followed by PGRN removal (Fig. 2A). On the other hand, administration of PGRN posterior to incubation with TNF-α also inhibited the TNF-α-induced upregulation of RhoA (Fig. 2B).
Fig. 2.Inhibition of the tumor necrosis factor-α (TNF-α)-induced upregulation of RhoA by recombinant progranulin (PGRN) in cultured human bronchial smooth muscle cells. A: effects of removal of pre-incubated PGRN. After a starvation period, the cells were incubated with PGRN (100 ng/ml) or PBS (vehicle for PGRN) for 1 h and were then washed by replacing with fresh serum-free medium. Subsequently, the cells were incubated with PGRN (100 ng/ml), TNF-α (30 ng/ml), and/or PBS (vehicle for PGRN and TNF-α), as indicated in panels at left, for 24 h. B: effects of posttreatment with PGRN. After a starvation period, the cells were treated with TNF-α (30 ng/ml) or PBS (vehicle for TNF-α). One hour after the treatment, the cells were treated with PGRN (100 ng/ml) or PBS (vehicle for PGRN) without washing out, and were incubated for 24 h. The expression levels of RhoA were determined by immunoblottings. Results are presented as means ± SE from six independent experiments. *P < 0.05 by one-way ANOVA with post hoc Dunnett.
Inhibition of the antigen-induced BSM hyperresponsiveness by PGRN in mice.
Next, effect of in vivo treatment with PGRN on variations of the airways induced by antigen exposure was determined in mice. Consistent with our previous studies (8, 13–15, 23), repeated OA inhalational challenge to the OA-sensitized mice caused an augmented contractility of the isolated BSM tissues to ACh (Fig. 3A, PBS-saline vs. PBS-OA groups). In this animal model of asthma, significant increases in RhoA protein expression (Fig. 3B, left) and BAL cell accumulation (Fig. 4A, left) were also observed as reported previously (8). In the sensitized control animals that received saline inhalation instead of antigen challenge, intranasal administrations of recombinant PGRN (100 ng/animal, 1 h before each saline inhalation) had no significant effect on ACh responsiveness and RhoA protein expression in BSMs and cell counts in BAL fluids (Fig. 3, A and B, and Fig. 4A, PBS-saline vs. PGRN-Saline groups). However, as shown in Fig. 3A, the intranasal administrations of PGRN significantly inhibited the BSM hyperresponsiveness to ACh induced by antigen exposure: the ACh concentration-response curve was significantly shifted to downward by the PGRN treatments (Fig. 3A, PBS-OA vs. PGRN-OA groups, P < 0.001 by two-way ANOVA). In addition, the RhoA upregulation in BSMs induced by antigen exposure was also inhibited significantly by the PGRN treatments (Fig. 3B, PBS-OA vs. PGRN-OA groups, P < 0.05). In the ACh (10−3 M)-stimulated BSMs, the increase in active form of RhoA observed in the antigen-challenged mice was attenuated by the PGRN treatments (Fig. 3C). On the other hand, the PGRN treatments did not affect significantly on the antigen-induced inflammatory cell accumulation in BAL fluids (Fig. 4A, PBS-OA vs. PGRN-OA groups). Similar results were also obtained by histochemical analyses of lung sections (Fig. 4, B–D).
Fig. 3.Effects of in vivo treatment with recombinant progranulin (PGRN) on ovalbumin antigen (OA)-induced bronchial smooth muscle (BSM) hyperresponsiveness (A) and upregulations of total amount (B) and active form (C) of RhoA protein in BSMs of mice. Male BALB/c mice were actively sensitized and repeatedly challenged with OA as described in methods. Animals also received intranasal administration of PGRN (100 ng/animal) or its vehicle PBS 1 h before each OA inhalational challenge. Twenty-four hours after the last OA challenge, measurement of BSM responsiveness to acetylcholine (ACh) (A) and expression levels of total amount of RhoA in BSMs (B) were performed as described in methods. Results are presented as means ± SE from four to eight independent experiment. Statistical significance was analyzed by two-way ANOVA (A) or one-way ANOVA with post hoc Dunnett (B). *P < 0.05 vs. PBS-saline, and †P < 0.05 vs. PBS-OA groups. C: determination of active form of RhoA by a pull-down assay. The freshly isolated BSMs of respective groups of mice were incubated for 10 min in the absence (–) or presence (+) of 10−3 M ACh, and the pull-down assays were performed as described in methods. Representative blots for total RhoA (input) and active RhoA (pulled down) are shown in panels at top. The bands for active RhoA were analyzed by a densitometer and densities of the bands are summarized in the panels at bottom. Results are presented as means ± SE from three independent experiments. *P < 0.05 vs. the other groups by one-way ANOVA with post hoc Bonferroni.
Fig. 4.Effects of in vivo treatment with recombinant progranulin (PGRN) on ovalbumin antigen (OA)-induced airway inflammation in mice. Male BALB/c mice were actively sensitized and repeatedly challenged with OA as described in methods. Animals also received intranasal administration of PGRN (100 ng/animal) or its vehicle PBS 1 h before each OA inhalational challenge. Twenty-four hours after the last OA challenge, cell counts in bronchoalveolar lavage (BAL) fluids (A) and hematoxylin and eosin staining of lung sections (B–D) were performed. A: cell counts in BAL fluids. Results are presented as means ± SE from four to eight independent experiments, respectively. Statistical significance was analyzed by one-way ANOVA with post hoc Dunnett. *P < 0.05 vs. PBS-saline group. Typical hematoxylin and eosin stainings of lung sections from PBS-saline (B), PBS-OA (C), and PGRN-OA mice (D).
PGRN inhibited antigen-induced upregulation of Ccl2 mRNA in BSMs without affecting TNF-α levels in BAL fluids.
Reportedly, gene expression of monocyte chemotactic protein (MCP)-1 (Ccl2) is mediated by TNF-α/NF-κB signaling in airway smooth muscle cells (37). To determine the inhibitory effect of PGRN on TNF-α/NF-κB signaling in vivo, the levels of Ccl2 mRNA in BSM tissues and TNF-α in BAL fluids were monitored in the present study. As shown in Fig. 5A, the upregulation of Ccl2 mRNA in BSMs induced by antigen exposure was significantly inhibited by the intranasal administrations of PGRN. On the other hand, the PGRN treatments did not affect significantly on the antigen-induced increments of TNF-α levels in BAL fluids (Fig. 5B).
Fig. 5.Effects of in vivo treatment with recombinant progranulin (PGRN) on ovalbumin antigen (OA)-induced upregulations of Ccl2 mRNA in bronchial smooth muscle (BSM) tissues (A) and tumor necrosis factor-α (TNF-α) levels in bronchoalveolar lavage fluids (BALFs) (B) in mice. Male BALB/c mice were actively sensitized and repeatedly challenged with OA as described in methods. Animals also received intranasal administration of PGRN (100 ng/animal) or its vehicle PBS 1 h before each OA inhalational challenge. Twenty-four hours after the last OA challenge, Ccl2 mRNA levels in BSM tissues and TNF-α levels in BAL fluids were determined by quantitative real-time reverse transcription PCR (A) and enzyme-linked immunosorbent assay (B), respectively, as described in methods. Results are presented as means ± SE from five animals. Statistical significance was analyzed by one-way ANOVA with post hoc Dunnett. *P < 0.05 vs. PBS-saline (A) or respective sensitized control (SC) (B), and †P < 0.05 vs. PBS-OA groups (A).
Downregulation of PGRN in the airways of the repeatedly antigen-challenged mice.
Finally, the expression level of PGRN in the airways of the repeatedly antigen-challenged mice was compared with that of the sensitized control animals. As a result, both the expressions of PGRN protein in BAL fluids and PGRN mRNA in BSM tissues were significantly decreased 24 h after the last antigen challenge (Fig. 6).
Fig. 6.Downregulation of progranulin (PGRN) in the airways of mice allergic asthma. Results are presented as aligned dot plots. The ovalbumin (OA)-sensitized mice were challenged with aerosolized OA or its vehicle saline, as described in methods. Twenty-four hours after the last inhalational challenge, PGRN protein levels in bronchoalveolar lavage fluids (BALFs) and PGRN mRNA levels in bronchial tissues were determined by enzyme-linked immunosorbent assay (A) and quantitative real-time reverse transcription PCR (B), respectively, as described in methods. The horizontal lines indicate means ± SE from six to seven independent experiments. *P < 0.05 vs. saline group by unpaired Student’s t-test.
DISCUSSION
PGRN is a growth factor with multiple biological functions and has been suggested as an endogenous inhibitor of TNF-α-mediated signaling. We show here that PGRN blocked the TNF-α-induced upregulation of RhoA in hBSMCs (Figs. 1 and 2), and that pretreatments of PGRN by intranasal instillations into the airways inhibited the BSM hyperresponsiveness and RhoA upregulation induced by antigen exposure (Fig. 3). Moreover, PGRN inhibited the antigen-induced upregulation of Ccl2, one of the genes that the expression is reportedly regulated by TNF-α/NF-κB signaling in airway smooth muscle cells (37), without affecting TNF-α production (Fig. 5). Thus PGRN could inhibit the antigen-induced TNF-α-mediated alteration of the airway smooth muscle cells.
RhoA is a ubiquitous protein and its amino acid sequence is highly conserved among species (7), suggesting that RhoA is involved in key biological pathways. In smooth muscle cells including airways, activation of RhoA causes Ca2+ sensitization of the contraction by activating its downstream Rho-kinases (40). Increasing evidence suggests an enhanced RhoA/Rho-kinases-mediated Ca2+ sensitization of the contraction in BSMs of asthmatics (2, 31, 41). The signaling of RhoA/Rho-kinases has been proposed as a new target for asthma therapy (7, 21, 22, 33). Several reports, including the present study (Fig. 3B), demonstrated that RhoA protein expression is upregulated in smooth muscles of the airways in mouse (8, 13, 31, 32), rat (12), and guinea pig (41) models of allergic asthma. Our previous studies have also demonstrated that the upregulation of RhoA protein is correlated with increased active form of RhoA, elevated levels of phosphorylated myosin light chain (MLC) phosphatase (phospho-MYPT1), and phospho-MLC, and augmented contraction, when BSMs of the diseased animals were stimulated with contractile agonists (9, 10, 15). Although the mechanism is not fully understood, TNF-α, which is also elevated in the airways of asthmatics (3, 47, 48), has been identified as one of the key mediators for the induction of antigen-induced upregulation of RhoA (18, 24, 31). Activation of NF-κB is believed to induce upregulation of RhoA in airway smooth muscle cells stimulated with TNF-α (24, 31). It is thus possible that the TNF-α-mediated signaling in airway smooth muscle might be a therapeutic target for treatment of the AHR in asthmatics.
Evidence suggests that the biological effect of PGRN is mediated, at least in part, by blocking TNF-α binding to its receptors (43). Consistently, incubation of hBSMCs with recombinant PGRN inhibited both the activation of NF-κB and the upregulation of RhoA induced by TNF-α (Fig. 1). Currently, pretreatment of hBSMCs with PGRN followed by PGRN removal was inefficient to inhibit the effect of a second TNF-α treatment (Fig. 2A). In addition, posttreatment with PGRN was effective to inhibit the TNF-α-mediated response (Fig. 2B). It is thus possible that the effect of PGRN is also mediated by blocking TNF-α binding to its receptors in the BSM cells. Considering that the administration of recombinant PGRN significantly alleviated inflammatory responses in various disease models, such as rheumatoid arthritis (29, 43), inflammatory bowel diseases (50), and brain (28), renal (20, 51, 56), and lung injuries (25), and that TNF-α is elevated in the airways of bronchial asthma (3, 35, 49), the effect of recombinant PGRN was determined in mice with allergic asthma. Recombinant PGRN was administered into the airways by the intranasal instillations before each antigen challenge because of the rapid increase of TNF-α level in the airways after the antigen exposure (Fig. 5B). As a result, the BSM hyperresponsiveness induced by antigen exposure was significantly inhibited by the PGRN pretreatments (Fig. 3A). Its inhibitory effect might result from the prevention of RhoA upregulation induced by antigen challenge (Fig. 3B). The hypercontraction of smooth muscles of the airways is suggested as one of the causes of AHR in asthmatics (34, 36). Thus, increasing PGRN levels in the airways might have a therapeutic effect on the AHR, a common feature of allergic bronchial asthma. On the other hand, recombinant PGRN, at the dosage used, did not block the antigen-induced increase in cell counts in BAL fluids (Fig. 4A), indicating that the inhibitory effect of PGRN on BSM hyperresponsiveness is not due to the inhibition of airway inflammation induced by antigen exposure.
In the present study, contrary to our expectations, recombinant PGRN had no significant effect on the antigen-induced airway inflammation, determined by measuring BAL cell counts (Fig. 4A). Similar results were also observed in histochemical analyses of the lungs (Fig. 4, B–D). Our previous studies demonstrated that antigen exposure caused an eosinophilic inflammation, that is, an increase in eosinophils in BAL fluids and lung sections (5, 6, 8), in this mouse model of bronchial asthma. Thus PGRN might have an ability to inhibit inflammations other than the eosinophil-mediated inflammation. Indeed, PGRN could inhibit the inflammations mediated mainly by neutrophils (19, 25, 55, 56). Our previous study also demonstrated no neutrophil accumulation in the airways in this animal model of asthma (5, 8). Alternatively, mediators other than TNF-α might be largely involved in the allergic airway inflammation.
The present study revealed that the protein expression of PGRN in BAL fluids was significantly decreased in the antigen-challenged mice (Fig. 6A). This is the first study, to our knowledge, that demonstrates a downregulation of PGRN in the airways of experimental allergic asthma. The decreased expression of PGRN mRNA seems to be one of the causes of downregulation of PGRN (Fig. 6B). Similarly, downregulation of PGRN has been reported in the injured kidney and heart of a murine model of hyperhomocysteinemia (20). Decreased expression of PGRN has also been suggested as a risk factor for Alzheimer’s disease (38) and Parkinson’s disease (4). PGRN insufficiency and deficiency have been reported to associate with Gaucher’s disease in human and mice, respectively (27). PGRN deficiency also exacerbated arthritis induced by collagen (43) and lung injury induced by lipopolysaccharide (53) in mice. In addition, auto-antibodies against PGRN have been found in several diseases, such as rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel disease, and such antibodies were shown to promote a pro-inflammatory environment (44, 45). On the other hand, exogenously applied recombinant PGRN could ameliorate disease progression in animal models (43, 50). Currently, the intranasal administration of recombinant PGRN significantly inhibited BSM hyperresponsiveness (Fig. 3A). Thus, although further studies are required to uncover the mechanisms of action of PGRN, increasing PGRN levels might be a promising therapeutic for multiple disorders, including allergic asthma.
PGRN seems beneficial to the current OA-induced murine model of allergic asthma. However, extrapolations to other asthma models and/or different species need to be assessed. Moreover, a therapeutic benefit would require experimentation with PGRN administrated in an already established disease, that is, postallergen challenge.
In conclusion, the present study revealed that intranasal instillation of recombinant PGRN into the airways ameliorated BSM hyperresponsiveness induced by antigen exposure in mice. The exogenously applied PGRN acts on BSMs to antagonize TNF-α produced by the antigen stimulation and inhibits the upregulation of RhoA, resulting in an inhibition of the augmented BSM contractility. Considering its mechanism of action, PGRN might be useful as an asthma controller in clinical use for AHR treatment. In addition, considering its inefficiency on eosinophil recruitment into the airways, PGRN might be more effective for non-eosinophilic-driven asthma, such as neutrophil-dominant asthma.
GRANTS
Y. Chiba was partially supported by Grant-in-Aid for Scientific Research (C) Grant 23590118 and Grant 15K08248 from the Japan Society for the Promotion of Science (JSPS).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Y.C., M.H., H.K., and H.S. conceived and designed research; Y.C., S.D., R.S., W.S., and Y.Y. performed experiments; Y.C., S.D., R.S., W.S., and Y.Y. analyzed data; Y.C., S.D., R.S., and Y.Y. interpreted results of experiments; Y.C., S.D., and W.S. prepared figures; Y.C., M.H., H.K., and H.S. drafted manuscript; Y.C., M.H., H.K., and H.S. edited and revised manuscript; Y.C., S.D., R.S., W.S., Y.Y., M.H., H.K., and H.S. approved final version of manuscript.
ACKNOWLEDGMENTS
We thank Gen Tanoue, Kazuki Shimizu, Takafumi Nakabaru, and Kazuki Fujii for technical assistance.
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