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. 2010 Nov 1;11(1):154.
doi: 10.1186/1465-9921-11-154.

Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus, remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Jae Youn Cho  1 , Dae Jae Song, Alexa Pham, Peter Rosenthal, Marina Miller, Shanna Dayan, Taylor A Doherty, Ajit Varki, David H Broide
Affiliations

Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus, remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Jae Youn Cho et al. Respir Res. .

Abstract

Background: In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge.

Methods: Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice.

Results: Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice.

Conclusions: Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium.

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Figures

Figure 1
Figure 1
Levels of BAL, lung, blood, and bone marrow eosinophils in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. Eosinophils in bronchoalveolar lavage (BAL), blood, and bone marrow were quantitated in cytospin slides stained with Wright-Giemsa, whereas eosinophils in lung sections were quantitated by immunostaining with an anti-MBP Ab. Chronic OVA challenge in WT mice induced a significant increase in BAL eosinophils (p < 0.0001*)(Fig 1A), peribronchial eosinophils (p < 0.0001*)(Fig 1B), and blood eosinophils (p < 0.003*)(Fig 1C)(WT no OVA vs WT OVA). Levels of eosinophils in OVA challenged Siglec-F deficient mice were significantly increased compared to WT mice challenged with OVA in the BAL (p < 0.001#)(Fig 1A), lung (p < 0.04#)(Fig 1B), blood (p < 0.003#)(Fig 1C), and bone marrow (p < 0.001#)(Fig 1D)(n = 16 mice/group).
Figure 2
Figure 2
Levels of mucus expression in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. The level of mucus expression was quantitated in lung sections by PAS staining using a light microscope objective at 20× (Fig 2 A-E). Chronic OVA challenge in WT mice induced a significant increase in the number of PAS+ mucus cells (p = 0.0001*)(WT no OVA vs WT OVA)(Fig 2A; Fig 2B vs Fig 2D). Levels of mucus expression were significantly increased in OVA challenged Siglec-F deficient mice compared to OVA challenged WT mice (p = 0.0001#)(Fig 2A; Fig 2E vs Fig 2D)(n = 16 mice/group).
Figure 3
Figure 3
Levels of peribronchial fibrosis, TGF-β1+ cells, and LTC4 levels in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. Levels of peribronchial fibrosis were quantitated by assaying collagen levels in lungs (Fig 3A), as well as by quantitating the area of peribronchial trichrome staining by image analysis (Fig 3B). Levels of mediators of lung fibrosis were assessed by quantitating the number of peribronchial cells immunostaining positive for TGF-β1 (Fig 3C) as well as levels of LTC4 in BAL (Fig 3D) by Elisa. Chronic OVA challenge in WT mice induced a significant increase in lung collagen (p < 0.0001*)(Fig 3A), and the area of peribronchial trichrome staining (p < 0.0001*)(Fig 3B), the number of peribronchial TGF-β1+ cells (p < 0.0001*)(Fig 3C), and levels of BAL LTC4 (p < 0.02*)(WT no OVA vs WT OVA). Levels of lung collagen were significantly increased in OVA challenged Siglec-F deficient mice compared to WT mice challenged with OVA (p < 0.002#)(WT OVA vs Siglec-F OVA)(Fig 3A), as was the area of peribronchial trichrome staining (p < 0.01#)(Fig 3B), and the number of peribronchial TGF-β1+ cells (p < 0.001)(Fig 3C)(n = 16 mice/group).
Figure 4
Figure 4
Levels of peribronchial fibronectin in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. Lung sections were immunostained with an anti-fibronectin Ab and the area of peribronchial fibronectin immunostaining determined by image analysis using a light microscope objective at 20× (Figure 4 A-E). Chronic OVA challenge in WT mice induced a significant increase the area of peribronchial fibronectin immunostaining (p < 0.0001*)(Fig 4A; Fig 4B vs Fig 4D)(WT no OVA vs WT OVA). The area of peribronchial fibronectin immunostaining was also significantly increased in OVA challenged Siglec-F deficient compared to OVA challenged WT mice (p < 0.01#)(Fig 4A; Fig 4E vs Fig 4D)(n = 16 mice/group).
Figure 5
Figure 5
Levels of lung cytokines and chemokines in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. Levels of IL-5 (Fig 5A), IL-13 (Fig 5B), eotaxin-1 (Fig 5C), and RANTES (Fig 5D) were measured in lung by ELISA. Chronic OVA challenge in WT mice induced a significant increase in IL-5 (p < 0.03*)(Fig 5A), IL-13 (p < 0.04*)(Fig 5B), and eotaxin-1 (p < 0.01*)(Fig 5C), but not RANTES (p = ns)(Fig 5D). Levels of IL-5 (Fig 5A) and IL-13 (Fig 5B) were no different in OVA challenged Siglec-F deficient mice compared to WT mice challenged with OVA, while levels of eotaxin-1 (p < 0.04#) (Fig 5C) and RANTES (p < 0.02#)(Fig 5D) were increased in Siglec-F deficient mice compared to WT mice challenged with OVA (n = 16 mice/group).
Figure 6
Figure 6
Quantitation of Siglec-F ligands in airway epithelial cells and peribronchial inflammatory cells. Lung sections from non-OVA or chronic OVA challenged WT or Siglec-F deficient mice were immunostained with a Siglec-F-Fc or a control Fc. The number of peribronchial cells (Fig 6A), as well as the area of airway epithelial cells (Fig 6B) immunostaining positive for Siglec-F-Fc was quantitated by image analysis. OVA challenged WT mice had significantly increased numbers of peribronchial cells immunostaining positive with the Siglec-F-Fc (p = 0.0001*)(Fig 6A) and significantly increased levels of airway epithelial cell Siglec-F-Fc immunostaining compared to non-OVA challenged WT mice (p = 0.0001*)(Fig 6B). Siglec-F deficient mice challenged with OVA had significantly increased numbers of peribronchial cells immunostaining positive for Siglec-F-Fc compared to OVA challenged WT mice (p = 0.01#)(Fig 6A), whereas airway epithelial Siglec-F-Fc immunostaining was similar in OVA challenged Siglec-F deficient and WT mice (Fig 6B)(n = 16 mice/group).
Figure 7
Figure 7
Quantitation of Siglec-F ligands in airway epithelial cells and peribronchial inflammatory cells in WT and Siglec-F deficient mice challenged with IL-4, IL-13 or TNF-α. WT or Siglec-F deficient mice were administered either IL-4, IL-13, TNF-α, or PBS diluent control. Twenty four hours after each individual cytokine or diluent challenge, the mice were sacrificed. BAL was obtained for determination of eosinophil and neutrophil cell counts, and the lungs were processed for immunohistology to detect Siglec-F ligand expression and MBP+ peribronchial eosinophils using a light microscope objective at 20×. Administration of either IL-4 (p < 0.001, WT; p < 0.001, Siglec-F deficient) or IL-13 (p < 0.001, WT; p < 0.001, Siglec-F deficient) induced a similar significant increase in levels of Siglec-F ligand expression by peribronchial cells (Fig 7A-C, 7E). In contrast, administration of TNF-α induced a small increase in peribronchial Siglec-F ligands (p < 0.01)(Fig 7E), but did not significantly increase Siglec-F ligand expression by airway epithelium in either WT or Siglec-F deficient mice (p = ns) (Fig 7 D, 7F). Administration of either IL-4 (p < 0.001 WT; p < 0.001 Siglec-F deficient) or IL-13 (p < 0.001 WT; p < 0.001 Siglec-F deficient) induced significantly increased levels of Siglec-F ligand expression by airway epithelium (Fig 7A-C, 7F). Although both IL-4 and IL-13 induced strong upregulation of Siglec-F ligand expression by airway epithelium (Fig 7F), levels of Siglec-F ligands in airway epithelium were slightly lower in Siglec-F deficient vs WT mice induced by IL-13 (p = 0.02) but not IL-4 (p = 0.10) (Fig 7F). IL-4 induced the strongest eosinophil response in BAL (WT p < 0.03; Siglec-F deficient p = 0.02)(Fig 7G) and lung (WT p < 0.001; Siglec-F deficient p < 0.001)(Fig 7H), while IL-13 and TNF-α induced a weaker eosinophil response in BAL (Fig 7G) and lung (Fig 7H). TNF-α, but not IL-4 or IL-13, induced a strong neutrophil response in BAL in both WT (p = 0.03) and Siglec-F deficient mice (p = 0.03) (Fig 7I)(n = 3 mice/group).
Figure 8
Figure 8
Thickness of the peribronchial smooth muscle layer and airway responsiveness in Siglec-F deficient vs WT mice. Different groups of Siglec-F deficient or WT mice were subjected to chronic OVA challenge. Non-OVA challenged mice served as a control. The thickness of the peribronchial smooth muscle layer was quantitated in lung sections (Fig 8A). Chronic OVA challenge in WT mice induced a significant increase in the thickness of the peribronchial smooth muscle layer (p = 0.0001*)(Fig 8A)(WT no OVA vs WT OVA). The thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice was significantly increased compared to WT mice challenged with OVA (p = 0.003#)(Fig 6A). Airway resistance (Raw) was measured (cm H2O.s/ml) in different groups of intubated and ventilated Siglec-F deficient deficient or WT mice following nebulization of either PBS diluent or MCh (3, 24, 48 mg/ml)(Fig 8B). Chronic OVA challenge in WT mice induced a significant increase in airway resistance (WT no OVA vs WT OVA; p < 0.002, 48 mg/ml MCh)(Fig 6B). Siglec-F deficient mice challenged with OVA had a statistically insignificant trend for increased airway responsiveness compared to WT mice (Siglec-F deficient OVA vs WT OVA; p = 0.15, 48 mg/ml MCh)(n = 16 mice/group).
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