Study design
As described in Fig. 1, after the initial sensitization, guinea pigs were intermittently exposed to aerosolized ovalbumin via up to three antigenic challenges. At the last challenge, the development of antigen-induced airway responsiveness was evaluated by generating dose-response curves showing the response to histamine before and after an antigenic challenge. Afterward, the animals were sacrificed to obtain lung and trachea samples. In the lung samples, areas in the lamina propria and the ASM layer were analysed through light microscopy. ASM cells were disaggregated from the trachea, and flow cytometry was used to determine the percentages of cells that expressed TGF-β1, IL-13 and SERCA2b. SERCA2B gene expression was determined in isolated ASM cells by RT-PCR. The level of GSH in ASM cells was measured by ELISA. Statistical analysis of the association of each parameter in ASM cells with values related to extrinsic and intrinsic airway obstruction was performed using Spearman correlation coefficient analysis. Some ASM cells were loaded with Fura-2 acetoxymethyl ester (Fura-2/AM), and the sarcoplasmic reticulum Ca2+ refilling rate after caffeine withdrawal was evaluated by microfluorometry. Control animals received sham treatment with saline solution.
Animals
The experiments were performed with healthy male guinea pigs with weights of 350-400 g that were originally purchased from Harlan Mexico (strain HsdPoc:DH) and bred in our institutional laboratory animal facilities. The guinea pigs were bred in a filtered, air-conditioned environment at 21±1°C with 50-70% humidity and 12/12-h light/dark cycles, fed sterilised pellets (2040 Harlan Teklad Guinea-Pig Diet; USA) and allowed access to water ad libitum.
Asthma model
Guinea pigs were sensitized and challenged with ovalbumin (asthma model) or saline (control) solutions according to previously described methods [17] (Fig. 1). Antigen or saline nebulization was performed in acrylic chambers, and pulmonary function was recorded by using a whole-body single-chamber plethysmograph for freely moving animals (Buxco Electronics Inc., Troy, NY, USA). The signal from the chamber was processed with the included software (Buxco Bio System XA v1.1) to calculate several respiratory parameters, including the broncho-obstructive index (Bi). This index was determined using the following equation:
Bi = ((Te-Rt)/Rt) (PEP/PIP)
where Te = expiratory time (s), Rt = relaxation time (s), PEP = peak expiratory pressure (cmH2O), and PIP = peak inspiratory pressure (cmH2O).
The software was adjusted to include only breaths with a tidal volume of 1 ml or more in the analysis, with a minimal inspiratory time of 0.15 s, a maximal inspiratory time of 3 s, and a maximal difference between the inspiratory and expiratory volumes of 10%. After the guinea pig was placed inside the plethysmographic chamber, a 5 min baseline Bi recording was initiated 10 min later. Starting one minute after aerosol administration, the Bi was recorded at 5 and 10 min and then every 15 min thereafter. Because the Bi was calculated for each breath, adjustments were made in the software to determine the average values for all breaths occurring over a 15 s interval and then to average these values during the last 5 min of each period.
Aerosols containing ovalbumin or saline were produced by a US-1 Bennett nebulizer (flow, 2 ml/min; Multistage liquid impinger, Burkard Manufacturing Co., Rickmansworth, Hertfordshire, UK), which released a mixture of particles with sizes of <4 µm (44%), 4-10 µm (38%), and >10 µm (18%).
Airway responsiveness was evaluated at the third antigenic challenge by the administration of non-cumulative doses of histamine (0.001 to 0.1 mg/ml) aerosol before and after antigen or saline exposure. The histamine doses were delivered over 1 min, and the Bi over the following 10 min was determined. The interval between histamine doses was 10 min. The dose-response curve was complete when the Bi reached three times its baseline level. The second histamine curve was determined three hours after the antigen or saline challenge.
Isolation of tracheal smooth muscle cells
One hour after completing the second histamine curve, guinea pigs were subjected to an overdose of an intraperitoneal injection of pentobarbital sodium (65 mg/kg), and the trachea and left lung lobe were obtained. Airway smooth muscle that was free of epithelium and connective tissue was dissected and incubated for 10 min at 37°C in 5 ml of Hanks’ solution (Gibco, Grand Island, NY, USA) containing 2 mg of cysteine and 0.05 U/ml papain. The tissue was washed in Leibovitz's solution and placed in physiological saline solution containing (mM): 25 NaHCO3, 118 NaCl, 1.2 KH2PO4, 4.6 KCl, 1.2 MgSO4, and 11 glucose. The smooth muscle was cut into small strips (5 x 0.5 mm) weighing 200 mg total and placed in 2.5 ml physiological saline solution containing collagenase type I (1 mg/ml; Boehringer-Mannheim, Indianapolis, IN, USA) and dispase II (4 mg/ml) for 10 min at 37°C. This procedure was repeated twice. Leibovitz's solution was added to stop the enzymatic activity, and the tissue was dispersed mechanically until isolated cells were observed.
Flow cytometry
Isolated smooth muscle cells were incubated with 10 g/ml brefeldin-A for 4 h at 37°C to inhibit cytokine release. Then, the cells were fixed with 4% p-formaldehyde for 10 min at 4°C, washed, and permeabilized with 0.1% saponin, 10% BSA and 1% NaN3 in PBS. The cells then underwent gentle shaking in the dark for 15 min at room temperature and were labelled with surface marker antibodies (1 µl/1 x 106 cells) against SERCA2b (clone 2A7-A1, mouse monoclonal SERCA2b ATPase, Abcam, Cambridge, MA, USA), IL-13 (human monoclonal, labeled with APC; BD Biosciences, San Diego, CA, USA) and TGF-b1 (clone 9016, labeled with phycoerythrin; R&D Systems, Minneapolis, MN, USA). Another incubation was performed with the secondary antibody conjugated to FITC (BD Biosciences Pharmingen, San Diego, CA, USA) for 30 min. Then, the cells were analysed for marker expression with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest software, and 10,000 events were counted. To analyse the staining, the blasts were first gated according to their physical properties (forward and side scatter). Next, a second gate was drawn based on the fluorescence characteristics of the gated cells, and the fluorescence intensity was assessed with histograms. The intensity of the fluorescence staining is expressed as the mean fluorescence intensity. Control staining was performed using fluorochrome-conjugated isotype-matched antibodies. Background staining contributed to <1% of the total signal and was subtracted from the experimental values.
Real-time quantitative polymerase chain reaction
Total RNA was extracted from tracheal smooth muscle strips obtained from guinea pigs using TRIzol reagent (Life Technologies, Grand Island, NY, USA). The RNA quality was assessed by resolving the fragments on denatured 1% agarose gels and measurement of the absorbance ratios at 260/280 nm. Total RNA (1 mg) was reverse-transcribed using Moloney murine leukaemia virus reverse transcriptase and 2 μg of random primers according to the manufacturer’s protocol (Advantage RT-for-PCR Kit; Clontech, Palo Alto, CA, USA). Quantitative real-time PCR amplification was performed using the iCycler iQ Detection System (Bio-Rad, Hercules, CA, USA). PCR was performed with the cDNA working mixture in a 20 μl reaction volume containing 20 mM Tris-HCl, 2 μl of cDNA, 200 μM dNTP, 2 mM MgCl2, 50 mM KCl, 1 μM each of the specific 5’ and 3’ primers, 1.25 U of Taq DNA polymerase (Roche, Branchburg, NJ, USA) SYBR green (1:50,000) and 10 nM fluorescein (Roche, Indianapolis, IN, USA) at pH 8.3. The primer pair used for SERCA2b amplification was designed in Primer BLAST. The sequences were 5’- TTAAAGCAACTGTCTATTTCTGCTG-3’ and 5’- AGTCAGAAAAAGCAAAACAAAATCTA-3’ (Merck KGaA, Darmstadt, Germany). The PCR cycling conditions consisted of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The 18S gene was used as an endogenous gene to normalize the RNA expression (Applied Biosystems® Eukaryotic 18S rRNA Endogenous Control (FAM™ MGB Probe, Non-Primer Limited), Thermo Fisher Scientific, USA). After 40 cycles, the delta-Ct method was used to compare the levels of the transcripts. The results were calculated as 2 elevated to the negative power of the difference between the CT value of each gene minus the CT value of 18S (2-Dct).
Measurement of intracellular Ca2+
Myocytes were loaded with 3 µM Fura-2/AM in physiological saline solution for 1 h at 25ºC. Then, the cells were allowed to settle into a perfusion chamber mounted on a Nikon inverted microscope (Diaphot 200; Minato-ku, Tokyo, Japan). The cells that adhered to the glass were continuously perfused at a rate of 2.5 ml/min with physiological saline solution (37°C, equilibrated with 95% O2 and 5% CO2, pH 7.4) containing 2 mM Ca2+. Cells were excited by alternating pulses of 340- and 380-nm-wavelength light, and the light emitted at 510 nm was measured using a PTI microphotometer (Photon Technology International, Princeton, NJ, USA). The cells in the field were removed before beginning the experiments. The background fluorescence was automatically subtracted by removing cells from the field. The fluorescence acquisition rate was 0.5 s. The intracellular Ca2+ concentration was calculated according to the Grynkiewicz et al. [21] formula. The Kd of Fura-2 was assumed to be 386 nM [22]. The mean 340/380 fluorescence ratios corresponding to Rmax and Rmin were determined by exposing the cells to 10 mM Ca2+ in the presence of 10 µM ionomycin and to Ca2+-free phosphate saline solution with 1.11 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N`,N`-tetraacetic acid (EGTA), respectively. Rmax was 11.7, and Rmin was 0.5. The fluorescence ratio resulting from 380 nm light excitation in Ca2+-free solution and Ca2+-saturated cells (ß) was 7.5.
Reduced glutathione (GSH) measurement
Isolated myocytes were rapidly homogenized, and 100 mg of pelleted cells were mixed with 300 μL of 5% saline solution. The samples were centrifuged at 12,000 x g at 4°C for 20 min. The supernatant was collected, and quantitative measurement of GSH in the supernatant was performed with the GSH assay kit (ab235670, Abcam, USA). This kit utilizes a specific enzymatic cycling method in the presence of GSH and a fluorophore. The reduction of the fluorophore produces a stable fluorescent product, the fluorescence of which is directly proportional to the amount of GSH in the sample and can be tracked kinetically (Ex/Em=535/587 nm).
Automated morphometry analysis in lung tissues
The left lung lobe was fixed by manually perfusing it with 10% neutral buffered formaldehyde solution. The lung fragments were cut and embedded in paraffin, and 4 µm-thick lung sections were stained with Masson trichrome stain. The areas containing lamina propria and smooth muscle (µm2) were determined through the use of automated morphometry (Qwin, Leica Microsystems Imaging Solutions, Cambridge, UK). All measurements were conducted in six bronchi and six bronchioles chosen at random from each animal. The data were adjusted according to the length of the corresponding basement membrane, and their average value was considered the final result. Bronchi and bronchioles were identified by the presence or absence of cartilage in the airway wall, respectively.
Drugs and reagents
Ovalbumin (chicken egg albumin grade II), Fura-2/AM, brefeldin-A, caffeine, cyclopiazonic acid, EGTA, ionomycin, papain, dispase II, cysteine, histamine dihydrochloride, and all salts and stains used for microscopy were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aluminium hydroxide was purchased from J.T. Baker (Phillipsburg, NJ, USA). Pentobarbital sodium was acquired from Pfizer (Toluca, Mexico). Fura-2/AM and CPA were dissolved in dimethyl sulfoxide (final concentration 0.025%). In the control experiments, dimethyl sulfoxide had no effect.
Statistical analysis
The Dbaseline Bi represented the difference between the reinforcement value and the last baseline Bi value obtained before challenge. Airway responsiveness to histamine was evaluated according to the provocative dose 200% (PD200), i.e., the interpolated histamine dose that caused a three-fold increase in comparison to the basal Bi. Changes in histamine responsiveness induced by saline or antigenic challenges were evaluated by means of a paired Student’s t-test and by dividing the PD200 observed after challenge by the PD200 value observed before challenge (PD200 ratio). For multiple group comparisons, repeated measures ANOVA followed by Dunnett’s tests was used. Associations between TGF-β1, GSH or SERCA-2b and changes in lung function were assessed through Spearman correlation coefficient analysis. Statistical significance was indicated by a two-tailed P < 0.05. The data in the text and figures are expressed as the mean ± SEM.