Allergen sensitization and exposure
Male Dunkin Hartley guinea pigs (initial weight 200-250 g) were obtained from HB Sahlins Försöksdjurfarm, Malmö, Sweden. Guinea pigs were sensitized by intradermal injection of a suspension of 3, 1, and 0.1 mg of TMA in 0.1 ml corn oil on day 1, 10 and 20, respectively (Figure 1). Intradermal injection (i.d.) of corn oil was used as control substance in non-sensitized animals. In double sensitization experiments, animals were sensitized as above but also administered 0.3 mg ovalbumin in 0.1 ml PBS i.d. On day 30 to 34, animals were exposed to an aerosol of TMA conjugated to guinea pig serum albumin (TMA-GPSA). Guinea pigs were exposed either to a high dose (0.15% TMA-GPSA) once for 15 min (day 34), or to low doses (0.03% TMA-GPSA) for 15 min each day on 5 consecutive days (day 30-34), in a Plexiglas chamber (Figure 1). Aerosol of the vehicle (PBS) was used as control. The aerosols were generated by a compressed air jet nebulizer (Maxin MA2, Clinova Medical AB, Malmö, Sweden; mean mass diameter < 3 μm in water). Pyrilamine (10 mg/kg, i.p.) was used 30 min before aerosol exposure to block the effects of histamine to minimize the potential fatal airway responses. This study was approved by the animal ethics committee in Göteborg. Dnr 88/98.
Study design
Animals were divided into four groups (7-12 in each group). Group 1 was not sensitized (i.d. corn oil), and was sham-exposed to PBS on 5 consecutive days. Group 2, 3, and 4 were sensitized to TMA. Group 2 was exposed to PBS on 5 consecutive days; group 3 was exposed to PBS on 4 consecutive days, followed by a single high dose of TMA-GPSA (0.15%) exposure; group 4 was exposed to low dose of TMA-GPSA (0.03%) on 5 consecutive days. The total dose of allergen received by animals of group 3 and 4 was exactly the same. ACh responsiveness was evaluated 24 hours after the last exposure. The early airway response to TMA-GPSA was measured one hour after the completion of the ACh responsiveness. BAL fluid was taken after the early airway response measurement, for evaluation of inflammatory cells and levels of LTC4/D4/E4, TXB2 and PGE2 (Figure 1).
Cholinergic airway responsiveness
Anaesthetized and ventilated guinea pigs were provoked with inhaled aerosol of acetylcholine (ACh) (1 × 10-5, 3 × 10-5, 1 × 10-4 and 3 × 10-4 M; 10 ml/kg for 20 inhalations) at a 10-min interval until RL increased at least 500% above the baseline. Aerosol was generated by an ultrasonic nebulizer (Pulmo-Sonic Model 2511, The DeVilbiss Co., Somerset, PA, U.S.A.) and driven by a ventilator. Lung resistance (RL) was measured as previously described [22] immediately after each ACh provocation for 10 min. A dose-response curve was constructed, and cholinergic airway responsiveness was expressed as provocative concentration of ACh, which increased RL 500% above the baseline (-Log PC500). Ten minutes before ACh provocation, animals were pretreated with pyrilamine (5 mg/kg, i.v.), propranolol (1 mg/kg, i.v), and suxamethonium 5 mg i.v.).
Early phase airway response to allergen
One hour after the completion of ACh assessment, animals were challenged with intratracheal instillation of 250 μg TMA-GPSA in 50 μl PBS. Lung resistance was measured for 10 min. The immediate airway response was expressed as area under the curve (AUC) of RL from 0 to 10 minutes for statistical analysis.
Specificity of early phase airway response to exposed allergen
To evaluate the specificity of early phase airway response to exposed allergen, we sensitized animals of three groups (7 in each group) with both TMA and OVA. Group 1 was exposed to PBS, group 2 to 0.03% TMA-GPSA, and group 3 to 0.03% OVA on five consecutive days. Early phase airway response and Evans blue dye exudation to OVA challenge were evaluated 24 hours after the last exposure.
Analysis of Evans blue dye exudation
Evans Blue dye (20 mg/kg) was injected i.v. over one minute, two minutes before allergen challenge. Animals were sacrificed by an over-dose of anaesthesia after the assessment of airway obstruction. The thoracic cavity was opened and systemic and pulmonary circulation was perfused with 100 ml PBS, respectively. The trachea, main bronchi, proximal intrapulmonary airways (PIA), distal intrapulmonary airways (DIA) was assessed. All tissues were freeze dried (MicroModulyo, Edwards High Vacuum International, West Sussex, U.K.) for 24 hours and then weighted. Evans Blue dye was extracted in 2 ml formamide in a 37°C water bath for 16 hours. Absorption of extracted Evans Blue dye at 620 nm was measured with a spectrophotometer (PU 8670 VIS/NIR, Philips, Norden, Stockholm, Sweden). The concentration of Evans Blue dye was quantified by interpolation on a standard curve and expressed as ng dye/mg dry tissue. The Evans Blue dye measurement has previously been shown to highly correlate with the exudation of radio labeled albumin in guinea pig airways [25].
Effects of LT1 receptor and COX on early phase airway response
Three groups of guinea pigs (8-11 in each group) were sensitized with TMA. Ten days after sensitization, we evaluated the roles of LT1 receptor and COX on the early phase airway response and plasma exudation to TMA-GPSA challenge by applying ICI 198,615 (a LT1 receptor antagonist) and indomethacin (a non-specific COX-inhibitor). In this experiment, animals of group 1 were pretreated with the vehicle for ICI 198,615 and the vehicle for indomethacin 10 minutes before TMA-GPSA challenge. Group 2 received ICI 198,615 (10-6 mol/kg, i.v.) and the vehicle for indomethacin, and group 3 received ICI 198,615 (10-6 mol/kg, i.v.) and indomethacin (10 mg/kg, i.v.). All animals were injected with Evans blue dye, two minutes before the intratracheal instillation of TMA-GPSA. The early phase airway response and Evans blue dye exudation were evaluated [25].
Inflammatory cells in BAL fluid
After the assessment of early phase airway response, animals were immediately sacrificed by over-dose of anaesthesia and the bronchial alveolar lavage (BAL) fluid was taken. For performing this, the thoracic cavity was opened. The systemic and pulmonary circulation were perfused through the left ventricle and the pulmonary artery with 100 ml PBS without Ca2+ and Mg2+, pH7.4, at room temperature to remove blood in the circulation. The airways were lavaged through the tracheal cannula by gentle instillation and aspiration of sterile PBS 4 ml for 5 times at room temperature. One ml BAL fluid was centrifuged at 3000 rpm at 4°C for 10 min, supernatant was collected and kept at -80°C until LTC4/D4/E4, TXB2, and PGE2 analysis. Total cell number in BAL fluid was determined in a haemocytometer using Türks solution. Cytocentrifuged preparations were stained with Giemsa and differential cell counts were carried out on 300 cells according to standard morphologic criteria.
Measurement of LTC4/D4/E4, TXB2 and PGE2
LTC4/D4/E4, TXB2 and PGE2 in BAL fluid and airway tissue culture medium were measured with commercial enzyme immunoassay (EIA) kits (Cayman Chemical Company, MI, USA) according to the manufacturer's instructions. The concentrations of LTC4/D4/E4, TXB2 and PGE2 in BAL fluid are expressed as pg/ml BAL fluid. The productions of LTC4/D4/E4 and TXB2 in the airway tissue in vitro are dependent on the size of the airway tissue and incubation time. To standardize the measurement, the production of LTC4/D4/E4, TXB2 was therefore expressed as pg/mg protein of the airway tissue/min of incubation time. The volume of protein in the airway tissue was measured with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, CA, USA).
Capacity of airway to produce cysLTs and TXB2
Animals of two groups (8 in each group) were sensitized with TMA. One group was exposed to PBS and one group was exposed to 0.03% TMA-GPSA on five consecutive days. Animals were sacrificed by over-dose of anaesthesia twenty-four hours after the last exposure, The thoracic cavity was opened, pulmonary and systemic circulations were perfused with PBS. Trachea, main bronchi, and intrapulmonary airways were dissected from each other and each portion was cut into 10 aliquots respectively. The airway tissue mixtures were made by randomly taking one aliquot from each portion. After equilibration by incubation in 1 ml culture medium (Waymouth MB 752/1) at 37°C for 2 hours, airway tissue was washed by removing the medium and replacing it with 1 ml fresh medium for four times. The tissue was then incubated for additional 15 min in 1 ml fresh medium complemented with either (1) 10 μl of the vehicle for calcium ionophore (DMSO, final concentration 0.2%) and 10 μl of the vehicle for arachidonic acid (AA) (95% ethanol); (2) 10 μl calcium ionophore (A23187) (final concentration 30 μM) and 10 μl of the vehicle for arachidonic acid (AA); and (3) 10 μl AA (final concentration is 10, 30, 100 μM) and 10 μl of the vehicle for A23187. The culture medium and airway tissue were collected and kept at -80°C until analysis of LTC4/D4/E4 and TXB2.
Activity of protein tyrosine kinase Lyn after repeated allergen exposure
Animals of two groups (10 in each group) were sensitized with TMA. Group 1 was exposed to PBS and group 2 to 0.03% TMA-GPSA on five consecutive days. Twenty-four hours after the last exposure, we isolated the airway tissues and measured the activity of enzyme protein tyrosine kinase Lyn in the airway tissues with ELISA. Activity of protein tyrosine kinase lyn in the homogenate of the airway tissue was measured with a commercial ELISA kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. The production of phosphopeptides in the tissue homogenate is dependent on the activity and amount of the protein tyrosine kinase as well as incubation time. To standardize the measurement, the activity of protein tyrosine kinase Lyn was therefore expressed as pg phosphopeptides/mg protein of tissue homogenate/min of incubation time.
Drugs and chemicals
The following drugs and chemicals were used: ketamine hydrochloride (Park-Davis S.A., Barcelona, Spain); xylazine chloride (Bayer Sverige AB, Göteborg, Sweden); suxamethonium chloride (KabiVitum AB, Stockholm, Sweden); propranolol, pyrilamine, acetylcholine, corn oil, Waymouth MB 752/1 medium, calcium ionophore (A23187), DMSO, arachidonic acid and ovalbumin (Sigma-Aldrich Chemical Co., St Louis, USA); ICI 198,615 (Zeneca AB Sweden); indomethacin (A/S Dumex, Danmark); trimellitic anhydride (Sigma-Aldrich) and TMA-GPSA conjugate (Dept. of Occupational and Environmental Medicine, Lund University Hospital, Sweden).
Data and statistical analysis
Data are expressed as mean ± S.E.M. for each group. Mann-Whitney U-test was used to test statistical significance of difference between two groups. If three or more groups were involved, Kruskal-Wallis test was used at first to ascertain that significant variance exists among the groups studied. A p-value less than 0.05 was considered significant.