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Inhibition of Neutrophil Elastase Attenuates AHR and Airway Inflammation 6 Hr After Secondary Challenge
Mice sensitized and challenged (primary) and mice challenged only were re-challenged (secondary) with OVA. Those mice which had previously been sensitized and challenged with OVA and treated with vehicle developed AHR compared to the non-sensitized but OVA challenged and re-challenged mice. When mice were treated with sivelestat, AHR were significantly reduced compared to vehicle saline-treated mice (Figure 1A). In parallel, inflammatory cell recruitment into the airways was increased 6 hrs after secondary airway challenge of previously sensitized and challenged animals (Figure 1B). Increased total cell numbers were largely due to increased numbers of neutrophils in BAL fluid (47% of total BAL fluid cells). When mice were treated with sivelestat, the numbers of eosinophils and lymphocytes were decreased significantly compared with vehicle-treated mice.
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Figure 1.
Neutrophil elastase inhibitor attenuates AHR 6 hr after secondary challenge. (A) Changes in RL 6 hr after secondary challenge. RL values to increasing concentrations of inhaled MCh were measured in non-sensitized/OVA-challenged mice receiving saline (PBS/OVA/vehicle/OVA), OVA-sensitized/OVA-challenged mice receiving saline (OVA/OVA/vehicle/OVA), and OVA-sensitized/OVA-challenged mice receiving sivelestat (OVA/OVA/Sivelestat/OVA). Results for each group are expressed as the mean ± SD. (n = 16–24 in each group). (B) Cell composition in BAL fluid obtained 6 hr after secondary challenge. Results for each group are expressed as the mean ± SD. (n = 16 in each group). *Significant differences (P<0.05) between PBS/OVA/OVA/vehicle and OVA/OVA/OVA/vehicle. #Significant differences (P < 0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
Lung Inflammation 6 Hr After Secondary Challenge
In previous studies, the development of AHR was associated with inflammatory changes in lung tissue. To determine if sivelestat affected inflammatory changes in the lung, we assessed tissue inflammation 6 hr after secondary OVA challenge. Hematoxylin-eosin and anti-Gr-1 monoclonal antibody stained lung tissue showed significant increases of neutrophil and lymphocyte numbers in peribronchial inflammation in previously sensitized and challenged animals compared to the non-sensitized mice. Mice treated with sivelestat demonstrated reduced the numbers of lymphocytes in lung tissue (Figure 2A, 2B, 2C).
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Figure 2.
Treatment with sivelestat reduces airway inflammation 6 hr after secondary challenge. (A) H&E–stained lung tissue (final magnification:x400). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (B) Inflammatory cell numbers in the peribronchial and perivascular tissue were measured (final magnification:x1000). (C) Anti-Gr-1 monoclonal antibody stained lung tissue (final magnification:x400). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (D) Treatment with sivelestat suppresses goblet cell metaplasia. PAS staining was performed to identify mucus-containing cells in the airway epithelium (final magnification:x1000). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (E) The number of mucus-positive cells. Data represent the mean ± SD. (n = 8 in each group). *Significant differences (P<0.05) between PBS/OVA/vehicle/OVA and OVA/OVA/vehicle/OVA. #Significant differences (P<0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
Lung sections were stained with PAS to identify mucus-containing cells in the airway epithelium (Figure 2D). A significant increase in numbers of PAS positive cells was found in previously sensitized and challenged mice compared with non-sensitized but re-challenged mice. Treatment with sivelestat significantly reduced the number of PAS positive cells per millimeter of basement membrane (Figure 2E).
Cytokines, Chemokines and Growth Factor Levels in BAL Fluid 6 Hr After Secondary Challenge
Six hr after secondary allergen challenge, BAL fluid was obtained to assess cytokine and chemokine levels. After secondary challenge, Th2 (IL-4, IL-5 and IL-13) cytokines were all increased in sensitized and challenged mice treated with vehicle compared to non-sensitized mice. Treatment with sivelestat significantly reduced the levels of IL-4, IL-5 and IL-13 (Figure 3). Levels of eotaxin, KC, and MIP-2 in BAL fluid were also increased in sensitized and challenged mice treated with saline compared to non-sensitized mice, and treatment with Sivelestat significantly reduced the level of eotaxin but not KC or MIP-2 (Figure 3).
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Figure 3.
Treatment with sivelestat alters cytokine, chemokine, and growth factor levels in BAL fluid 6 hr after secondary challenge. The levels of (A) IL-4, (B) IL-5, (C) IL-13, (D) TGF-β1, (E) Eotaxin, (F) KC, and (G) MIP-2 in BAL fluid were measured. The results for each group are expressed as the mean ± SD. (n = 16 in each group). *Significant differences (P < 0.05) between PBS/OVA/vehicle/OVA and OVA/OVA/vehicle/OVA. #Significant differences (P < 0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
Inhibition of Neutrophil Elastase Prior to Secondary Challenge Attenuates Lung Allergic Responses 48 Hr After Secondary Challenge
We previously showed that at 48 hr after secondary allergen challenge, the inflammatory reaction and AHR developing after primary challenge resolved but that the re-challenge induced a strong inflammatory reaction with development of AHR. Indeed, as observed with the increases in AHR 6 hr after the secondary challenge, these increases in AHR 48 hr after the secondary challenge were also significant (Figure 4A). Under these conditions, treatment with sivelestat significantly prevented the increases in AHR.
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Figure 4.
Treatment with sivelestat reduces AHR and airway inflammation 48 hr after secondary challenge. (A) Changes in RL 48 hr after secondary challenge. Results for each group are expressed as the mean ± SD. (n = 16 in each group). (B) Cell composition in BAL fluid. Results for each group are expressed as the mean ± SD. (n = 16 in each group). (C) H&E–stained lung tissue (final magnification:x400). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (D) Inflammatory cell numbers in the peribronchial and perivascular tissue (final magnification:x1000). (E) Anti-Gr-1 monoclonal antibody stained lung tissue (final magnification:x400). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (F) PAS staining (final magnification:x1000). (a) PBS/OVA/vehicle/OVA, (b) OVA/OVA/vehicle/OVA, (c) OVA/OVA/Sivelestat/OVA. (G) The number of mucus-positive cells. Data represent the mean ± SD. (n = 8 in each group). *Significant differences (P<0.05) between PBS/OVA/vehicle/OVA and OVA/OVA/vehicle/OVA. #Significant differences (P < 0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
At 48 hr, increased total cell numbers were largely due to increased numbers of eosinophils and lymphocytes in the recovered BAL fluid. Administration of sivelestat at the time of the secondary challenge led to a significant decrease in eosinophil numbers in BAL fluid (Figure 4B). Sensitized and challenged mice treated with vehicle showed remarkable accumulation of the numbers of eosinophils and lymphocytes in lung tissue at 48 hr and administration of sivelestat significantly reduced the numbers of eosinophils and lymphocytes in lung tissue (Figure 4C, 4D, 4E) as well as numbers of goblet cells (Figure 4F, 4G).
Forty-eight hr after the secondary challenge, IL-13 and IL-5 levels were increased in sensitized and challenged mice treated with vehicle compared to non-sensitized mice. Treatment with sivelestat significantly reduced IL-13 levels in BAL fluid. Levels of TGF-β1, and MIP-2 in BAL fluid were also increased in sensitized and challenged mice treated with vehicle compared to non-sensitized mice, and treatment with sivelestat significantly reduced the levels of TGF-β1 (Figure 5).
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Figure 5.
Treatment with sivelestat alters cytokine and growth factor levels in BAL fluid 48 hr after secondary challenge. The levels of (A) IL-4, (B) IL-5, (C) IL-13, (D) TGF-β1, (E) Eotaxin, (F) KC, and (G) MIP-2 in BAL fluid were measured. The results for each group are expressed as the mean ± SD. (n = 16 in each group). *Significant differences (P<0.05) between PBS/OVA/vehicle/OVA and OVA/OVA/vehicle/OVA. #Significant differences (P < 0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
Serum Anti-OVA IgE Antibody Levels After Secondary Challenge
6 hr and 48 hr after secondary allergen challenge, serum was obtained to assess OVA-specific IgE levels. Levels of OVA-specific IgE were significantly increased in sensitized and challenged mice treated with vehicle compared with non-sensitized but challenged mice. Treatment with sivelestat did not affect serum OVA-specific IgE levels, likely since initial sensitization and challenge were completed before administration of the inhibitor (Table 1).
PAR-2 Expression in Lung Tissue
PAR-2 has been reported to be one of the receptors for neutrophil elastase, and is expressed on a variety of cells including airway epithelial cells, fibroblasts, myocytes, sensory neurons, and bronchial and vascular smooth muscle. PAR-2 was detected intracellularly in eosinophils but at undetectable levels on the cell surface. However, once these few receptors became activated, PAR-2 was redistributed from intracellular stores to the surface of the cell. In lung tissue assessed for PAR-2 staining, non-sensitized and challenged mice showed few PAR-2 positive cells (Figure 6A(a), 6A(b), 6B), whereas sensitized and challenged and both 6 and 48 hr after secondary allergen challenged mice showed increased numbers of PAR-2 positive cells (Figure 6A(c), 6A(e), 6B). Treatment with sivelestat did not affect the number of PAR-2 positive cells (Figure 6A(d), 6A(f), 6B).
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Figure 6.
PAR-2 expression on lung tissue and T cells in PBLN. (A) PAR-2 stained lung tissue obtained 6hr and 48 hr after secondary challenge (final magnification:x400). (a) 6 hr: PBS/OVA/vehicle/OVA, (b) 48 hr:PBS/OVA/vehicle/OVA, (c) 6 hr: OVA/OVA/vehicle/OVA, (d) 6 hr:OVA/OVA/Sivelestat/OVA, (e) 48 hr: OVA/OVA/vehicle/OVA, (f) 48 hr: OVA/OVA/Sivelestat/OVA. (B) The number of PAR-2-positive cells. *Significant differences (P<0.05) between PBS/OVA/vehicle/OVA and OVA/OVA/vehicle/OVA. Data represent the mean ± SD (n = 6 in each group). (C) Dotted line: Control antibody, Thin line: anti-human PAR-2 mAb, Non/Non/Non, Bold line: anti-human PAR-2 mAb, OVA/OVA/OVA. PAR-2 expression of CD3 and CD4 T Cells in PBLN. (a) CD3 T cells in the PBLN and (b) CD4 T Cells in PBLN were assessed by intracellular staining. The data shown are representative of three independent experiments. Increased numbers of PAR-2 positive CD3 and CD4 T cells from the PBLN of the sensitized and challenged mice were observed.
PAR-2 Expression on PBLN T Cells
To determine the expression of PAR-2 in T cells, percentages of PAR-2-positive CD3 and CD4 T cells in PBLN were assessed by intracellular staining. As shown in Figure 6C, increased numbers of PAR-2 positive CD3 and CD4 T cells were observed in the PBLN of sensitized and challenged and secondary challenged mice. Sivelestat did not have any significant effect on the number of PAR-2 positive CD3 and CD4 T cells in the PBLN (data not shown).
Effect of Neutrophil Elastase Inhibition on in Vitro Cytokine Production From Spleen Cells
To determine whether the difference in cytokine levels observed in the BAL fluid of mice treated with sivelestat were due to a difference in antigen-specific T-cell responsiveness, spleen cells were isolated 6 hr after secondary OVA challenge, and re-stimulated in culture for 24 hrs with OVA. There were no significant differences in cultures of cells from mice treated with sivelestat and those treated with saline in for IFN-γ production. After culture with OVA, spleen cells from mice treated with sivelestat secreted significantly lower amounts of IL-13 than did spleen cells from mice treated with vehicle (Figure 7).
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Figure 7.
Effect of neutrophil elastase inhibitor for in vitro cytokines production. In vitro cytokine production from spleen cells obtained 6 h after secondary challenge. The levels of (a) IL-4, (b) IL-5, (c) IL-13 and (d) IFN-γ in culture supernatant of spleen cells from mice after OVA sensitization and challenge were measured. The results for each group are expressed as the mean ± SD. (n = 12 in each group). *P < 0.05 without re-stimulation with OVA groups (medium) vs. with re-stimulation with OVA groups. #Significant differences (P < 0.05) between OVA/OVA/vehicle/OVA and OVA/OVA/Sivelestat/OVA.
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