A2B adenosine receptor expression by myeloid cells is proinflammatory in murine allergic-airway inflammation

Abstract

Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Because of its role in regulating myeloid cell-dependent inflammatory processes, we examined A(2B) R expression by myeloid cells in a cockroach allergen model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A(2B) R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and proinflammatory cytokine secretion after final allergen challenge in sensitized mice. A(2B) R deficiency resulted in a dramatic reduction on Th2-type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A(2B) R dependent. In contrast, there were no differences in the levels of the CXC chemokines keratinocyte-derived chemokine and MIP-2 in the myeloid cell A(2B) R-deficient mice, strengthening A(2B) R involvement in the development of Th2-type airways inflammation. Proinflammatory TNF-α, IFN-γ, and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A(2B) R deletion mouse lines. Our results demonstrate Th2-type predominance for A(2B) R expression by myeloid cells as a mechanism of development of asthma-like pulmonary inflammation.

Figure 1. Airways mucus production

(A) PAS stain in lung histological sections of WT, A_2BR KO and LysM KO groups of mice. Each panel is a representative image, the mucin is stained magenta within the airway epithelial cells. (B) Quantization of the total area of PAS stained mucus using ImageJ photoanalysis software. A_2BR mice showed significant decreases in airways mucus production as compared to WT. Values are mean ± SEM of 2 independent experiments for WT (n = 5), A_2BR KO (n = 6) and LysM KO (n= 6). p values are indicated on the figure.

Figure 2. Pulmonary eosinophil recruitment and infiltration

Lung homogenate (LH) concentrations of eotaxin 1 (Panel A) and 2 (Panel B) in WT (n = 5), A_2BR KO (n = 6) and LysM KO (n = 6) mice. (C) Representative images of parenchymal eosinophils in H+E sections of whole left lung. (D) LH supernatant EPO activity in WT, A_2BR KO and LysM KO mice. (E) Representative cytospin photomicrographs of BAL cells including eosinophils from the indicated mice. (F) Absolute eosinophil counts in the BAL fluid. The A_2BR deficient groups of mice demonstrated markedly reduced eosinophil infiltration into the BAL and lung parenchyma and concurrent diminished production of eosinophil chemoattractants. Values are the mean ± SEM of two independent experiments in panels A, B, D, and F. For panels D and F, n = 9 for WT, n = 10 for A_2BR KO and n = 10 for LysM. p values are indicated on the figure.

Figure 3. Pulmonary neutrophil infiltration

Absolute counts of neutrophils recovered from the BAL fluid (A). Lung homogenate supernatant MPO activity (B). No significant differences were observed in the infiltration of neutrophils into the airspaces or lung parenchyma between the experimental groups. Values are the mean ± SEM of two independent experiments with n = 9 for WT, n = 10 for A_2BR KO and n = 10 for LysM KO. Lung homogenate concentrations of KC (C), MIP-2 (D) and RANTES (D) in WT, A_2BR KO and LysM KO groups of mice. Values are mean ± SEM of WT (n = 5), A_2BR KO (n = 6) and LysM KO (n = 6) for panels C, D, and E. p values are indicated on the figure.

Figure 4. Pulmonary macrophage and lymphocyte infiltration

Absolute counts of macrophages (A) and lymphocytes (B) recovered in the BAL fluid of WT, A_2BR KO and LysM KO groups of mice. Macrophages recovered in BAL trended towards decreased but were only significantly depressed in the LysM KO mice. In contrast, lymphocyte levels were markedly reduced in the BAL fluid of A_2BR deficient groups of mice. Values are the mean ± SEM of two independent experiments with n = 9 for WT, n = 10 for A_2BR KO and n = 10 for LysM KO. p values are indicated on the figure.

Figure 5. Th2 and proinflammatory/immunomodulatory cytokines

Lung homogenate (LH) concentrations of the Th2 cytokines IL-4 (A), IL-5 (B) and IL-13 (C) all demonstrated a significant decrease in the Th2 cytokines. Additionally, the proinflammatory cytokines IL-17 (D) and TNF-α (E) were decreased in the A_2BR and LysM KO mice. The classic Th1 cytokine, IFN-γ, was also significantly decreased in the KO groups (F). Values are the mean ± SEM of two independent experiments with WT (n = 5), A_2BR KO (n = 6) and LysM KO (n = 6). p values are indicated on the figure.