Prostaglandin E(2) exerts homeostatic regulation of pulmonary vascular remodeling in allergic airway inflammation

Abstract

Nonselective inhibition of PG synthesis augments inflammation in mouse models of airway disease, but the roles of individual PGs are not completely clarified. To investigate the role of PGE(2) in a mouse model of airway inflammation induced by a natural allergen, we used mice lacking the critical terminal synthetic enzyme, microsomal PGE(2) synthase (mPGES)-1. Mice lacking mPGES-1 (ptges(-/-) mice) and wild-type C57BL/6 controls were challenged intranasally with low doses of an extract derived from the house dust mite Dermatophagoides farinae (Der f). The levels of PGE(2) in the bronchoalveolar lavage fluids of Der f-treated ptges(-/-) mice were approximately 80% lower than the levels in wild-type controls. Der f-induced bronchovascular eosinophilia was modestly enhanced in the ptges(-/-) mice. Both Der f-treated strains showed similar increases in serum IgE and IgG1, as well as comparable levels of Th1, Th2, and Th17 cytokine production by Der f-stimulated spleen cells. These findings indicated that mPGES-1-derived PGE(2) was not required for allergen sensitization or development of effector T cell responses. Unexpectedly, the numbers of vascular smooth muscle cells and the thickness of intrapulmonary vessels were both markedly increased in the Der f-treated ptges(-/-) mice. These vascular changes were suppressed by the administration of the stable PGE(2) analog 16, 16-dimethyl PGE(2), or of selective agonists of the E-prostanoid (EP) 1, EP2, and EP3 receptors, respectively, for PGE(2). Thus, mPGES-1 and its product, PGE(2), protect the pulmonary vasculature from remodeling during allergen-induced pulmonary inflammation, and these effects may be mediated by more than one EP receptor.

FIGURE 1

Effect of Der f challenge on PG generation by pulmonary tissues of ptges^−/− mice and WT C57BL/6 controls. Mice were treated intranasally twice weekly for 3 wk with the indicated doses of Der f. Fortyeight hours after the last challenge, the mice were euthanized and BAL fluid was collected. Lungs were mechanically dispersed and incubated in the presence of arachidonic acid. A, PGE₂ levels in BAL fluids (detected by ELISA) after the lipid fractions were purified. Data are the mean ± SD from four to six mice per group. The experiment was performed three times. B, GC-MS analysis of all PGs generated by lung homogenates from the indicated mouse strains after 30 min of incubation in the presence of arachidonic acid (50 μM). The supernatants were processed for GC-MS as detailed in Materials and Methods. Results depicted are mean ± SD from five mice/group.

FIGURE 2

Parameters of the immune response to Der f in ptges^−/− mice and WT C57BL/6 controls. A, Total IgE (top) and IgG1 (bottom) in serum from mice treated with saline or Der f were measured by ELISA. Results are the mean 6 SD from 10–12 mice/group in three separate experiments. B, Cytokine generation by restimulated spleen cells. Spleens were harvested from mice 48 h after the final Der f challenge. The cells were mechanically dispersed and passed through nylon mesh strainers before being stimulated for 72 h with Der f (20 μg/ml) in medium. The content of cytokines was determined by ELISA. Results are the mean ± SEM from 8–10 mice per group from two separate experiments.

FIGURE 3

Accumulation of leukocytes in the BAL fluid from ptges^−/− mice and WT C57BL/6 controls. The lungs of the saline- or Der f-treated mice were lavaged three times with 0.7 ml PBS/5 mM EDTA. Slides were prepared by cytocentrifugation and stained with Difquick. A, Total cell counts. B, Eosinophils expressed as total cell numbers (left) and as a percentage (right). C, Neutrophils expressed as total cell numbers (left) and as a percentage (right). The results are the mean ± SEM of 30–40 animals per group in 8–10 experiments.

FIGURE 4

Histologic analysis of pulmonary inflammation in Der f-treated mice and effect of 16, 16-dimethyl PGE₂. A, Images of H&E-stained lungs from the indicated groups of mice (original magnification ×200). B, Perivascular cellular infiltrates showing eosinophils stained with Congo red dye (original magnification ×630). C, PAS-stained lung showing goblet cells (original magnification ×200). Results in (A–C) are from single mice. D, Quantitative analysis of Der f-induced BV inflammation (left) and goblet cell metaplasia (right) and the effect of 16, 16-dimethyl PGE₂. Results are the mean ± SEM from 32–47 mice/group from 6–10 independent experiments.

FIGURE 5

Histologic and immunohistochemical analysis of smooth muscle hyperplasia occurring in response to Der f. A, Representative lung sections from mice of the indicated genotypes treated with saline or Der f. Sections were stained with an Ab against α-SMA to visualize bronchial (open arrows) or vascular (closed arrows) smooth muscle. Original magnification ×200. B, Quantitative analysis of vascular SMC numbers (left) and thickness (right). The effect of 16, 16-dimethyl PGE₂ is shown. Results are the mean ± SEM from 30 mice per group. C, Double immunostaining with Abs against α-SMA (red) and PCNA (brown). Results are from an individual mouse representative of five. Original magnification ×200 (left), ×630 (right). Stains of spleen (left) and human tonsil (right) are shown as controls. Original magnification ×200. D, Immunofluorescence of frozen lung sections showing double staining for SMA (green) and vWF (red) as markers of smooth muscle and endothelium, respectively. Representative stains of lungs from saline (left) and Der f-treated (right) ptges^−/− mice are shown. Original magnification ×630.

FIGURE 6

Effects of treatment with selective EP receptor agonists on the histologic responses of ptges^−/− mice to Der f. A, PAS stains showing goblet cell metaplasia in response to Der f and the effects of the indicated EP receptor agonists. B, Quantitative analysis of inflammation (top, as assessed by hematoxalyn-azure-2-eosin stains of BVB) and goblet cells (bottom), showing the effects of the agonists for the indicated receptors. C, Hematoxalin-azure-2-eosin stains showing BVB of the same mice as in (A). Original magnification ×200 for both A and C. D, Quantitative analysis of vascular remodeling in ptges^−/− mice treated with agonists for the indicated receptors. SMC numbers (top) and thickness of the smooth muscle layer (bottom) are shown. Results are the mean of 11–25 mice/group from at least three experiments.