β2 adrenergic agonist attenuates house dust mite-induced allergic airway inflammation through dendritic cells

Two batches of house dust mite (HDM) extract from Dermatophagoides farina (Der f) were provided by ITEA Inc. (Tokyo, Japan) as a lyophilized preparation of milled mites. Formoterol (FORM) was provided by Astellas Pharma Inc. (Tokyo, Japan). ADR antagonists used were propranolol for β1 + β2, ICI118,551 for β2, CGP20712A for β1, prazosin for α₁ and yohimbine for α₂-ADR respectively (Sigma-Aldrich Co. LLC., St. Louis, MO). Phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, epinephrine, phorbol 12-myristate 13-acetate (PMA) and ionomycin were purchased from Sigma-Aldrich Co. LLC.

Female BALB/c mice (6–8 weeks old, Japan SLC Inc., Hamamatsu Japan) were maintained at the Saga University animal facility under specific pathogen free conditions. Animal experiments were undertaken following the guidelines for care and use of experimental animals of the Japanese Association for Laboratory Animals Science (1987) and were approved by the Saga University Animal Care and Use Committee.

Mice were inoculated intranasally with 25 μg HDM or vehicle on days 0, 7 and 14. Mice were challenged intranasally with 5 μg HDM on days 21, 22 and 23. On day 24, mice were euthanized by intraperitoneal injection of sodium pentobarbital. Serum, bronchoalveolar lavage (BAL) fluid and lung tissue were harvested for further analysis (Figure 1a). FORM (12.5 ng/animal) was administered subcutaneously three times per week. FORM was dissolved in ethanol 10 mg/ml as a central stock, then was diluted optimal concentration using PBS. On the day when HDM was given, FORM was administered 30 minutes before the HDM inoculation.

Blood and BAL fluid samples were collected as described previously [22]. Briefly, blood was collected by right ventricle puncture. Serum was collected by centrifugation of whole blood at 8,000 × g for 5 min at 4°C and stored at −80°C until needed. Then a 20-gauge tube was inserted into the trachea and the lungs were lavaged twice with 1.0 ml of saline. The cell suspension was centrifuged at 100 × g for 5 min at 4°C. The total number of cells was counted using a hemocytometer. Cytospin samples were prepared from the cell suspension. Cell differentiation was determined by counting at least 300 leukocytes in samples stained with Diff-Quik (Siemens, Germany).

Histological examination was performed as previously reported [23]. Lungs were fixed with 10% neutralized buffered formalin (Wako, Osaka, Japan) and embedded in paraffin. Lung sections were stained with Hematoxylin and Eosin (H&E), and periodic acid-Schiff (PAS) stains.

After bronchoalveolar lavage, the left lung was isolated and homogenized in 50 mM Tris buffered saline (pH 7.4) containing 1 mM EDTA, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM Na₃VO₄ and 1 mM NaF. The lung homogenates were centrifuged at 10,000 × g for 15 min, and then supernatants were collected and stored at −80°C until required.

Mice were inoculated intranasally with HDM or vehicle as described above on days 0, 7 and 14. A bronchial lymph node was removed on day 17, crushed gently and a single cell suspension prepared by passing through a 40 μm nylon mesh (BD Falcon, Franklin Lakes, NJ). Lymphocytes were stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 4 hours. After stimulation, supernatants were collected and stored at −80°C.

IL-4, IL-6, IL-10, IL-13, IL-5, IL-17 and IL-23 were measured using a Quantikine ELISA Kit (R&D systems Inc., Minneapolis, MN) according to the manufacturer’s instructions. All samples were measured in duplicate.

Bone marrow (BM) cells were isolated from BALB/c mice as previously reported [24]. BM cells were resuspended at 2.0 × 10⁶ cells/ml in RPMI1640 medium supplemented with 10% fetal calf serum (FCS). The cells were cultured in the presence of 10 ng/ml recombinant murine GM-CSF (R&D systems) at 37°C in a humidified atmosphere containing 5% CO₂ for 6 days. On day 6, cells were harvested. More than 95% of the cells were positively stained with CD11c, and were used as myeloid dendritic cells (BMDCs) in the following experiments. BMDCs were stimulated with 10 μg/ml HDM, with or without FORM or epinephrine, for 24 hours. BMDCs were pretreated with 1 μm of propranolol, ICI118,551, CGP20712A, prazosin or yohimbine. BMDC supernatants were collected and stored at −80°C.

As shown in Figure 1b, BMDCs were stimulated with 10 μg/ml HDM, with or without 100 pM FORM for 24 hours. BMDCs were collected and washed twice, and then 1.0 × 10⁶ BMDCs were administered to naïve BALB/c mice intranasally. On day 10, mice were challenged with 25 μg HDM. Twenty four hours later, BAL fluid and bronchial lymph nodes were collected as described above. Lymphocytes were stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for 4 hours. After stimulation, supernatants were collected and stored at −80°C.

To measure Der f-specific IgG, MaxiSorp plates (Nunc, Roskilde, Denmark) were coated with 0.25 μg/ml Der f (Seikagaku co. Tokyo). Plates were washed with 0.1 M phosphate-buffered saline (PBS) containing 0.05% Tween 20. Each well was filled with a blocking solution of 1% BSA (Sigma-Aldrich) in PBS, and incubated for 1 hour. After washing 3 times, 100 μl/well of serum was added and the plates incubated for 1 hour. After washing, IgG1 bound to the plate was detected using biotin-labeled rat anti-mouse IgG1 (Bio-Rad AbD Serotec Ltd, Oxford UK), HRP-streptavidin (Sigma-Aldrich), and 3,3’,5,5’-tetramethylbenzidine (TMB, Invitrogen, CA). The amount of specific antibody was expressed as laboratory units (LU), and samples were compared with a standard serum containing Der f-specific IgG1.

The data are shown as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used for multiple comparison of continuous variables. When there was a significant difference, the difference between each group was tested using Scheffe’s test. All tests were two-sided with a five percent level of significance.

We investigated the effect of the β2 adrenergic agonist, FORM, on HDM-induced airway inflammation. Mice were sensitized and challenged with HDM, which resulted in an increase of total cells, neutrophils, lymphocytes and eosinophils in BAL fluid. FORM treatment significantly suppressed BAL cell numbers (Figure 2a). Differences between the two groups were as follows; total number of cells; 16.3 ± 5.3 and 5.3 ± 2.4 ± 10⁴/ml (p < 0.01), neutrophils; 5.4 ± 2.9 and 1.4 ± 1.0 ± 10⁴/ml (p < 0.01), lymphocytes; 0.9 ± 0.8 and 0.5 ± 0.4 ± 10⁴/ml (p = 0.45), and eosinophils; 2.8 ± 1.6 and 0.5 ± 0.6 ± 10⁴/ml (p < 0.01) in HDM mice and FORM treated mice, respectively. Histological examination showed that inflammatory cell infiltration and goblet cell metaplasia were suppressed by FORM (Figure 2b). The concentrations of IL-4 and IL-17 in lung tissue were significantly decreased in FORM treated mice (Figure 2c, 2f). IL-4 concentration was 19.8 ± 2.9 and 9.1 ± 3.3 pg/ml (p < 0.01) and IL-17 was 143.6 ± 63.1 and 47.4 ± 33.5 pg/ml (p < 0.01) in HDM mice and FORM treated mice, respectively. There was no difference in the concentration of IL-13 (118.4 ± 61.4 pg/ml in FORM v.s. 168.9 ± 39.8 pg/ml in HDM ), IL-5 (17.8 ± 7.6 pg/ml in FORM v.s. 21.9 ± 6.5 pg/ml in HDM )in lung tissue respectively (Figure 2d, 2e). The titer of serum HDM specific IgG in FORM mice (920.7 ± 600.3 LU/ml) was similar to that in the serum of HDM mice (1,270.0 ± 920.5 LU/ml) (Figure 2g).

We next examined whether FORM attenuates the T cell response to HDM in regional lymph nodes. HDM treatment caused an increase in the concentration of IL-4, IL-13 and IL17 in supernatant of lymphocytes (Figure 3). Lymphocytes from FORM treated mice produced significantly less cytokines; IL-4; 104.7 ± 2.8 and 91.5 ± 3.9 pg/ml (p < 0.01), IL-13; 205.7 ± 14.3 and 103.6 ± 4.3 pg/ml (p < 0.01), IL-5; 37.4 ± 2.5 and 21.1 ± 3.0 pg/ml (p < 0.01), and IL-17, 94.7 ± 15.4 and 58.5 ± 2.2 pg/ml (p < 0.01), in HDM and HDM/FORM treated mice, respectively. These finding suggest that FORM attenuates the Th2 and Th17 responses to HDM in vivo.

To eliminate the possibility that FORM directly affects cytokine production from lymphocytes, lymph node cells harvested from HDM mice were stimulated with PMA and ionomycin in the presence of various concentrations of FORM in vitro. As shown in Table 1, FORM concentrations of up to 10,000 pM did not alter IL-4, IL-13 or IL-17 production from lymphocytes.

We hypothesized that the suppression of T cell responses by FORM in vivo was mediated by DCs. We therefore examined the effect of FORM on cytokine production by BMDCs in vitro. BMDCs produced IL-23 and IL-6 after HDM stimulation. IL-23 production was suppressed by both FORM and epinephrine in a dose dependent manner (Figure 4a), where the effect of FORM was 10,000 times stronger than epinephrine. IL-6 production was also suppressed by FORM, but not by epinephrine at concentrations of up to 10⁻⁷ M (Figure 4b). IL-10 production from DCs was enhanced by FORM and epinephrine in a dose dependent manner (Figure 4c).

We examined whether the effect of FORM is mediated specifically by ADR. To clarify which ADR contribute to the suppression of cytokine production by FORM, specific ADR antagonists used were propranolol for β1 + β2, ICI118,551 for β2, CGP20712A for β1, prazosin for β1 and yohimbine for β2-ADR respectively. IL-23 production from HDM-stimulated BMDCs was suppressed by FORM, 195.2 ± 38.6 pg/ml, but was significantly restored to 301.9 ± 1.8 pg/ml (Figure 5a) when propranolol was added. ICI118,551 also significantly restored IL-23 production to 355.5 ± 29.5 pg/ml, while CGP20712A did not. Propranolol and ICI118,551 restored IL-6 production suppressed by FORM, but CGP20712A did not (Figure 5b). Interestingly, IL-10 production from HDM-stimulated DCs enhanced by FORM, 151.3 ± 17.0 pg/ml, was significantly decreased in a β₂ ADR pathway dependent manner (Figure 5c).

We used epinephrine, a full agonist for all classes of ADR, to investigate whether the modulation of DC cytokine production is mediated specifically by β2 ADR. The suppression of IL-23 production by epinephrine, 137.4 ± 21.4 pg/ml, was restored, to 182.3 ± 10.7 pg/ml by propranolol, and to 248.0 ± 26.1 pg/ml by ICI118,551. IL-23 production could not be restored by CGP20712A (105.4 ± 11.6 pg/ml), prazosin (121.6 ± 5.5 pg/ml) or yohimbine (124.5 ± 4.5 pg/ml) (Figure 5d). The suppression of IL-6 production by 1 μm epinephrine, 12.3 ± 2.4 ng/ml, was also restored to 20.9 ± 2.3 ng/ml by propranolol, and to 22.6 ± 2.5 ng/ml by ICI118,551. IL-6 production could not be restored by CGP20712A (14.2 ± 1.2 ng/ml), prazosin (13.6 ± 0.7 ng/ml) or yohimbine (13.1 ± 0.6 ng/ml) (Figure 5e). On the contrary, enhanced IL-10 production by epinephrine, 172.0 ± 11.1 pg/ml, was significantly reduced to 92.8 ± 2.7 pg/ml by propranolol and to 86.1 ± 5.9 pg/ml by ICI118,551 (Figure 5f).

To examine whether the effect of FORM on HDM-induced airway inflammation in vivo depends on DCs, we performed adoptive transfer of HDM-pulsed BMDCs to naïve mice. When intranasal administration of HDM-pulsed DCs was followed by HDM challenge, the total number of cells (31.2 ± 13.5 ± 10⁴/ml), eosinophils (2.3 ± 1.4 ± 10⁴/ml) and neutrophils (13.1 ± 6.9 ± 10⁴/ml) in BAL fluids was significantly increased compared with mice given sham-pulsed DC and HDM challenge (Figure 6a). BAL cell counts in the latter group were total cells; 12.1 ± 6.3 ± 10⁴/ml, eosinophils; 0.4 ± 0.4 ± 10⁴/ml, and neutrophils; 3.6 ± 3.4 ± 10⁴/ml. When DCs pulsed with HDM in the presence of 100 pM FORM in vitro were administered to mice, the BAL cell numbers were significantly decreased in comparison to HDM group; total cells; 19.0 ± 3.3 ± 10⁴/ml (p < 0.05), eosinophils; 0.3 ± 0.3 ± 10⁴/ml (p < 0.05), and neutrophils; 5.0 ± 0.9 ± 10⁴/ml (p < 0.05), respectively (Figure 6a).

Mice given HDM-pulsed DCs and antigen challenge showed lymphocyte responses similar to mice directly sensitized with HDM. The concentration of IL-4, IL-13, IL-5 and IL-17 in lymph node cell culture was 82.1 ± 31.8 pg/ml, 108.9 ± 32.8 pg/ml, 19.3 ± 6.9 pg/ml, and 181.7 ± 79.6 pg/ml, in the HDM-pulsed DC group, and 35.9 ± 30.3 pg/ml, 63.0 ± 26.2 pg/ml, 13.7 ± 6.2 pg/ml, and 43.1 ± 24.6 pg/ml, in the sham-pulsed DC group, respectively (Figure 6b-e). Significant differences between the two groups were found for levels of IL-4 (p < 0.05), IL-13 (p < 0.05) and IL-17 (p < 0.05). When DCs were pulsed with HDM in the presence of FORM, the concentration of IL-4, IL-13, IL-5 and IL-17 in lymph node cell culture supernatants decreased to 45.6 ± 21.6 pg/ml, 61.4 ± 24.9 pg/ml, 17.3 ± 5.1 pg/ml, and 76.3 ± 56.2 pg/ml, respectively. The difference between the HDM group and the HDM/FORM group was significant for levels of IL-13 and IL-17 (p < 0.05).