Role of sphingosine kinase 1 in allergen-induced pulmonary vascular remodeling and hyperresponsiveness

Acute allergen-induced inflammation. Airway resistance (resaw; A), airway responsiveness to MCh infusion (B), Ppa (C), and pulmonary vascular responsiveness to infused U46619 (D) were monitored in isolated perfused and ventilated murine lungs. BAL fluid leukocytes were counted and analyzed by means of microscopic analysis (E). Values are shown as means ± SEMs (WT mice: n = 10-16 per group; SphK1^−/− mice: n = 8-9 per group). ^∗P < .05, ^∗∗P < .01, and ^∗∗∗P < .001 versus corresponding PBS/OVA or as indicated. #P < .05.

Chronic allergen-induced inflammation. Airway resistance (resaw; A), airway responsiveness to MCh infusion (B), Ppa (C), and pulmonary vascular responsiveness to infused U46619 (D) were monitored in isolated perfused and ventilated murine lungs. BAL fluid leukocytes were counted and analyzed by means of microscopic analysis (E). Values are shown as means ± SEMs (WT mice: n = 10 per group; SphK1^−/− mice: n = 7-9 per group). ##P < .01 and ^∗∗∗P < .001 versus corresponding PBS/OVA-treated group. &P < .05 versus the WT PBS/OVA-treated group).

Pulmonary cytokine production was quantified in BAL supernatants by using the cytokine multiplex assay. Values are shown as means ± SEMs (n = 5-8 per group). P/O, PBS/OVA; O/O, OVA/OVA. ^∗P < .05, ^∗∗P < .01, and ^∗∗∗P < .001. Dotted lines indicate the detection limit of the cytokine assay and are missing if all values were within the working range.

Chronic allergen-induced lung inflammation (A-F). Histochemistry of small pulmonary arteries (hematoxylin and eosin staining [Fig 4, A and B]) and double-labeling immunohistochemistry (α–smooth muscle actin [α-SMA] green/CD68 red [Fig 4, C-F]; α-SMA green/Ki67 red [Fig 4, G and H]) of intra-acinar arteries in lungs from SphK1^−/− mice (Fig 4, A and E) and WT mice (Fig 4, B and F) after OVA/OVA treatment are shown. Fig 4, C and D, show intra-acinar arteries of the SphK1^−/− and WT PBS/OVA-treated groups. Acute allergen-induced lung inflammation is shown in Fig 4, G and H. Ki67-immunoreactive nuclei were found in smooth muscle cells of the SphK1^−/− (Fig 4, G) and WT (Fig 4, H) OVA/OVA-treated groups. These photomicrographs are representative of 3 to 5 mice examined per group. Bars = 50 μm (Fig 4, A-F); 20 μm (Fig 4, G and H).

Chronic allergen-induced lung inflammation (n = 6-9 per group.). A, Quantitative morphometric analysis: volume density of vascular walls in small pulmonary arteries and intra-acinar arteries in SphK1^−/− and WT mice after PBS/OVA or OVA/OVA treatment. B, Regression analysis of the vascular responsiveness to U46619 and the volume density of small pulmonary arterial walls in SphK1^−/− and WT mice.

Immunohistochemistry (A-C), toluidine blue staining (D and E), and ultrastructural analysis (D′ and E′) of arteries from SphK1^−/− mice (Fig 6, C and D) showed the presence of α–smooth muscle actin–immunoreactive smooth muscle cells luminal to the internal elastic lamina (arrows in Fig 6, A and B). Fig 6, C, shows a different orientation of smooth muscle cells in the media and intimal layer. OVA/OVA (Fig 6, E and E′) but not PBS/OVA (Fig 6, D and D′) arteries of SphK1^−/− mice contained smooth muscle (sm) luminal to the internal elastic lamina (arrows). Note the increased thickness of the internal elastic lamina in chronic challenged arteries (Fig 6, E′). ec, Endothelial cells. These photomicrographs are representative for 3 to 5 mice examined per group. Bars: 50 μm (Fig 6, A-C); 5 μm (Fig 6, D′ and E′).

Effect of age on pulmonary vascular responsiveness to infused U46619 was analyzed in isolated perfused and ventilated lungs of 10-week-old (10 w), 14-week-old (14 w), and 18-week-old (18 w) SphK1^−/− and WT mice. Values are shown as means ± SEMs (n = 5-7 per group).

Quantitative RT-PCR analysis: A-C, acute allergen-induced lung inflammation; D, chronic allergen-induced lung inflammation. Acute allergen challenge led to upregulation of S1PP2 (Fig E2, A) and S1Ply (Fig E2, B) mRNA in WT mice but not in SphK1^−/− mice. Acute inflammation induced downregulation of SphK2 and S1P1 mRNA in lungs from SphK1^−/− mice (Fig E2, C), whereas chronic inflammation reduced S1P1 mRNA in lungs from SphK1^−/− mice (Fig E2, D). The data represent 5 to 7 lungs per group. ^∗P < .05, ^∗∗P < .01, and ^∗∗∗P < .001.

Ultrastructural analysis of WT (A and B) and SphK1^−/− (C and D) capillary endothelium showed the presence of enlarged luminal spaces between cells in OVA/OVA-treated (arrows in Fig E3, B and D) but not PBS/OVA-treated (Fig E3, A and C) arteries. These photomicrographs are representative of 3 to 5 mice examined per group. Bars = 200 nm.