Phosphodiesterase 4B is essential for T(H)2-cell function and development of airway hyperresponsiveness in allergic asthma

Abstract

Background: Cyclic AMP (cAMP) signaling modulates functions of inflammatory cells involved in the pathogenesis of asthma, and type 4 cAMP-specific phosphodiesterases (PDE4s) are essential components of this pathway. Induction of the PDE4 isoform PDE4B is necessary for Toll-like receptor signaling in monocytes and macrophages and is associated with T cell receptor/CD3 in T cells; however, its exact physiological function in the development of allergic asthma remains undefined.

Objectives: We investigated the role of PDE4B in the development of allergen-induced airway hyperresponsiveness (AHR) and T(H)2-driven inflammatory responses.

Methods: Wild-type and PDE4B(-/-) mice were sensitized and challenged with ovalbumin and AHR measured in response to inhaled methacholine. Airway inflammation was characterized by analyzing leukocyte infiltration and cytokine accumulation in the airways. Ovalbumin-stimulated cell proliferation and T(H)2 cytokine production were determined in cultured bronchial lymph node cells.

Results: Mice deficient in PDE4B do not develop AHR. This protective effect was associated with a significant decrease in eosinophils recruitment to the lungs and decreased T(H)2 cytokine levels in the bronchoalveolar lavage fluid. Defects in T-cell replication, T(H)2 cytokine production, and dendritic cell migration were evident in cells from the airway-draining lymph nodes. Conversely, accumulation of the T(H)1 cytokine IFN-γ was not affected in PDE4B(-/-) mice. Ablation of the orthologous PDE4 gene PDE4A has no impact on airway inflammation.

Conclusion: By relieving a cAMP-negative constraint, PDE4B plays an essential role in T(H)2-cell activation and dendritic cell recruitment during airway inflammation. These findings provide proof of concept that PDE4 inhibitors with PDE4B selectivity may have efficacy in asthma treatment.

FIG 1

AHR in OVA-sensitized PDE4B^+/+ and PDE4B^−/− mice. C57BL6/129Ola PDE4B^+/+ and PDE4B^−/− mice were immunized and challenged with OVA (50 µg) in alum intraperitoneally on days 0 and 14, and intranasally (50 µg OVA in 50 µL saline) on days 14, 25, 26, and 27. Control mice received alum intraperitoneally and normal saline intranasally. On day 28, AHR in response to increasing concentrations of methacholine was measured either from conscious mice using a whole-body plethysmograph and expressed as enhanced pause (Penh; A) or from anesthetized and c-annulated mice (B and C). In the latter case, R_L and C_dyn are reported. Data are the means ± SEMs with the number of mice in each group in parentheses. *P < .005 (compared with the OVA-sensitized wild-type littermates).

FIG 2

Airway inflammation in OVA-sensitized PDE4B^+/+ and PDE4B^−/− mice. Mice were sensitized as described in Fig 2 (A) or by reducing the intranasal challenge to 3 (on days 14, 25, and 26; B). After sensitization, the mice were anesthetized, and BAL was performed. Total and differential cell counts in the BALF were determined as detailed in the Methods. Data are the means ± SEMs (n = 13–19 mice/genotype in A, and N = 6–7 mice/genotype in B). *P < .05; **P < .01 (compared with OVA-sensitized PDE4B^+/+ mice). C, Levels of T_H2 cytokines (IL-4, IL-5, and IL-13) and IFN-γ in the BALF supernatants were determined by ELISA. Data are the means ± SEMs (5–6 mice/genotype) *P < .05; **P < .005. C, Control; Eos, eosinophils; Lym, lymphocytes; Mo, monocytes and macrophages.

FIG 3

Inflammatory cell infiltration and PAS-stained mucus-producing cells in the lungs of PDE4B^+/+ and PDE4B^−/− mice. Mice were immunized and challenged with OVA as described in Fig 2. One day after the last OVA challenge, lungs were processed for staining with PAS. Arrows point to goblet cells (magenta) in the airway epithelium (A). The quantitation of the degree of inflammation is reported in B (P < .05; N = 6–7).

FIG 4

T_H2-cell responses in OVA-sensitized PDE4B^+/+ and PDE4B^−/− mice. Mice were sensitized as described in Fig 2. Single-cell suspensions of the bronchial lymph nodes were prepared and cultured in the presence of increasing OVA concentrations (A) or in the presence of 30 µg/mL OVA alone, a combination of OVA and 10 µmol/L rolipram, or vehicle (control; C). Proliferation of OVA-specific cells was assessed by incorporation of [³H]thymidine over the last 16 hours of the 3-day culture. Data are the means ± SEMs (n = 12 mice/genotype in A and 4–7 mice/genotype in C). *P < .05 (compared with the wild-type cells treated with OVA alone). B, Cytokine levels in the culture medium collected after 4 days of incubation in the presence of 30 µg/mL OVA were determined by ELISA. Data are the means ± SEMs (n = 7–13 mice/genotype). *P < .01. Conc., Concentration; Rol, rolipram.

FIG 5

Proliferation of splenocytes and popliteal lymph node cells from OVA-primed PDE4B^+/+ and PDE4B^−/− mice. A, Mice were given 1 dose of OVA (50 µg) in alum intraperitoneally. Nine days later, the spleen was excised, and cells were cultured in the presence of increasing OVA concentrations. B, Mice were given OVA conjugated with incomplete Freund adjuvant by injection into hind footpads intradermally (75 µg OVA each footpad). Nine days later, popliteal lymph nodes were excised, and cells were cultured in the presence increasing OVA. Proliferation of OVA-specific cells was assessed by incorporation of [³H]thymidine over the last 16 hours of 3-day culture. Data are the means ± SEMs. *P < .05 (compared with the corresponding wild-type cells).

FIG 6

Dendritic cell migration from the lung to draining lymph nodes (LNs). Naive PDE4B^+/+ and PDE4B^−/− mice were administered FITC-OVA. After 24 hours, the bronchial and submaxillary lymph nodes were excised and a single-cell suspension prepared. Cells were stained with phycoerythrin-conjugated antimouse MHC II and APC-conjugated antimouse CD11c mAbs followed by fluorescence-activated cell sorting analysis. Representative histograms from 3 PDE4B^+/+ and PDE4B^−/− mice are shown in A. The percentage of FITC⁺ cells in a population of MHC-IIhⁱ, CD11c⁺ cells is summarized in B. Cells from submaxillary LNs were used as a negative control. *P < .05 compared with wild-type mice (N = 8 mice/group).