NPY and NPY receptors in airway structural and inflammatory cells in allergic asthma

Abstract

Purpose: Neuropeptide Y (NPY) level is elevated in allergic asthmatic airways and activation of NPY receptor-1 (NPY-Y1) on antigen-presenting cells (APCs) is essential for T cell priming. Paradoxically, NPY-Y1 modulates hyper-responsiveness in T cells, suggesting a bimodal role for NPY in APCs and T cells. Therefore, determination of the temporal and spatial expression pattern of NPY and its receptors in asthmatic airways is essential to further understand the role of NPY in allergic asthma.

Methods: Lungs were isolated from control and acute and chronic stages of OVA-sensitized and challenged mice (OVA). Stains, including H&E, PAS, and trichrome, were used to determine the severity of lung pathology. The expression patterns of NPY and NPY-Y receptors in the airways were determined using ELISA and immunofluorescence. Cytokine levels in the BALF were also measured.

Results: NPY levels were undetectable in the BALF of control mice, but significantly increased in the OVA group at day 80. Levels of IL-4, TGF-β1 and TGF-β2, significantly increased and peaked on day 45 and decreased on day 80 in the OVA group, exhibiting an inverse correlation with NPY levels. NPY expression was localized to macrophage-like cells in the peri-bronchial and peri-vascular areas in the lung tissue. NPY-Y1 and -Y5 receptors were constitutively expressed by both structural and inflammatory cells in the lung tissue.

Conclusions: NPY produced by activated macrophage-like cells may be involved in regulating cytokine production and cellular activities of immune cells in asthma. However, it remains unclear whether such an increase in NPY is a defensive/compensatory mechanism to modulate the effects of inflammatory cytokines.

Figure 1

(A) OVA antigen sensitization protocol; (B) AHR analysis: Graph showing the specific airway resistance (R_L) in tracheostomized mice in response to aerosolized methacholine in PBS-treated and OVA-sensitized and -challenged mice; (C) Bronchoalveolar lavage (BAL) fluid cell analysis: Showing graph of total and differential inflammatory cell count in BAL fluid of PBS and OVA-sensitized and -challenged mice. Data are shown as mean ± SEM of values of eight mice per group. *P<0.05; **P0.001.

Figure 2

(A) H&E staining: showing infiltration of inflammatory cells (arrow head) and hypertrophy of structural cells (arrow); (B) PAS staining: showing mucous cells (purple color) (red arrow); and (C) Trichrome staining: showing subepithelial collagen deposition (blue color stain) (yellow arrow) in the airway (AW) of PBS and of OVA-sensitized and challenged mice on days 33, 45 and 80. All pictures are representative of each staining technique. BAL fluid analysis: Showing graphs of differential (D) IL-4; (E) TGFβ1; (F) TGFβ2; and (G) NPY levels in BAL fluid using ELISA. Data are shown as mean ± SEM of values of five measurements in each group. *P<0.05; **P0.001.

Figure 3

(A) NPY expression in airway epithelial cells and smooth muscle cells: Confocal images (600×) showing immunofluorescent (IF) staining analysis of NPY (Cy3) in airway structural cells using epithelial cell adhesion molecule (EpCAM) (Cy2) as a selective marker for airway epithelial cells and α-actin as a specific marker for smooth muscle cells (Cy5). These are representative images of the IF staining. (B) NPY positive cell analysis: Graph showing number of NPY-positive (+) cells detected as part of the inflammatory cell population in the peri-bronchial area per high powered field (HPF). Data are shown as mean ± SEM of values of eight measurements in each group. *P<0.05; **P0.001.

Figure 3

(A) NPY expression in airway epithelial cells and smooth muscle cells: Confocal images (600×) showing immunofluorescent (IF) staining analysis of NPY (Cy3) in airway structural cells using epithelial cell adhesion molecule (EpCAM) (Cy2) as a selective marker for airway epithelial cells and α-actin as a specific marker for smooth muscle cells (Cy5). These are representative images of the IF staining. (B) NPY positive cell analysis: Graph showing number of NPY-positive (+) cells detected as part of the inflammatory cell population in the peri-bronchial area per high powered field (HPF). Data are shown as mean ± SEM of values of eight measurements in each group. *P<0.05; **P0.001.

Figure 4

NPY expression in macrophages: (A) Confocal images (600×) showing immunofluorescent (IF) staining analysis of NPY (Cy2) in inflammatory cells using F4/80 (Cy3) as a specific marker for macrophages. These are representative images of the IF staining. (B) F4/80 positive cell analysis: Graph showing number of F4/80-positive (+) cells detected as part of the inflammatory cell population in the peri-bronchial area per high powered field (HPF). (C) Percentage of NPY⁺ cells that are also positive for F4/80 (double positive) relative to the total number of F4/80⁺ cells. Data are shown as mean ± SEM of values of eight measurements in each group. *P<0.05; **P0.001.

Figure 5

NPY expression in APC-like cells: (A) Confocal images (600×) showing immunofluorescent (IF) analysis of NPY (Cy2) in inflammatory cells using MHC class II (Cy3) as a selective marker for APC-like cells. These are representative images of the IF staining. (B) MHC II positive cell analysis: Graph showing number of MHCII-positive (+) cells detected as part of the inflammatory cell population in the peri-bronchial area per high powered field (HPF). (C) Mean fluorescent intensity (MFI) in arbitrary units (using NIH Image J software) of MHCII expression in MHCII⁺ cells. (D) Percentage of NPY⁺ cells that are also positive for MHC II (double positive) relative to the total number of MHCII⁺ cells. Data are shown as mean ± SEM of values of eight measurements in each group. *P<0.05; **P0.001.

Figure 6

NPY receptor expression in airway: (A) Confocal images (600×) showing immunofluorescent (IF) staining analysis of (A) NPY receptor 1 (NPY1R) (Cy2) and (B) NPY receptor 5 (NPY5R) (Cy3) in the airway. These are representative images of the IF staining. These are representative images of the IF staining.