Table 1.
Primer sequences.
Fig 1.
Effects of rhIL-26 on STATs phosphorylation in lung tissue.
Mice received intranasal instillation of recombinant human (rh) IL-26 protein or its vechicle (PBS), with or without prior instillation of endotoxin (LPS) or its vehicle (PBS). The total and phosphorylated STAT1 (A) and STAT3 (B) proteins were measured with Western blot in lung tissues 24 hours (h) after the instillations (n = 10). Each lane of the bands represents a separate blot. Data are presented as mean ± SEM.
Fig 2.
Effects of rhIL-26 on the accumulation of BAL cells and cytokines.
Mice received intranasal instillation of recombinant human (rh) IL-26 protein or its vechicle (PBS), with or without prior instillation of endotoxin (LPS) or its vehicle (PBS). Bronchoalveolar lavage (BAL) samples were harvested 6 hours (h) (n = 8), 24 h (n = 10) and 72 h (n = 8) after the instillations. The number of (A) total leukocytes and differential cellularity including (B) neutrophils, (C) macrophages and lymphocytes (S1 Table) were determined. Cytokines were measured using Luminex™ in the cell-free BAL fluid for (D) IL-6, (E) CCL2, (F) CCL3 and (G) G-CSF. Correlation between concentrations of (H) CCL2 protein and macrophages in 6 h BAL samples and between concentrations of (I) CCL3 and neutrophils in 72 h BAL samples were also calculated based upon the acquired data. Data are presented as mean ± SEM.
Fig 3.
Effects of rhIL-26 on cytokine mRNA in lung tissue.
Mice received intranasal instillation of recombinant human (rh) IL-26 protein or its vechicle (PBS), with or without prior instillation of endotoxin (LPS) or its vehicle (PBS). Lung tissue samples were harvested 6 h (n = 8), 24 h (n = 10) and 72 h (n = 8) after the instillations. The harvested tissue was assessed for messenger (m) RNA of cytokines using RT-qPCR. Data shown includes (A) IL-6, (B) TNF-α, (C) CCL20, (D) CCL2, (E) CXCL1, (F) CXCL2 and (G) G-CSF. Data are presented as mean ± SEM.
Fig 4.
Effects of rhIL-26 on lung tissue inflammation and MPO protein content.
Mice received intranasal instillation of recombinant human (rh) IL-26 protein or its vechicle (PBS), with or without prior instillation of endotoxin (LPS) or its vehicle (PBS). Lung tissue samples were harvested 6 h (n = 8), 24 h (n = 10) and 72 h (n = 8) instillations. Histological evaluations were performed on lung tissue sections stained with hematoxylin and eosin (H&E) and quantified with a scoring system. Representative H&E-staining pictures (magnification: 10×, scale bar: 50 μm) are shown (A). The inflammation in four tissue compartments (the interstitial area, alveolar space, peribronchial area and perivascular area) on lung tissue sections was evaluated and scored (see Methods section) at (B) 6 h, (C) 24 h, and (D) 72 h after the intranasal instillation, respectively. The MPO protein in lung tissue was analyzed using immunoblot. The bands of each mouse and the densitometry ratio of MPO to actin are shown in (E). Each panel relates to a time-point and each lane of the bands represents a separate blot. (F) The MPO content in cell-free BAL fluid samples was quantified using ELISA. Data are presented as mean ± SEM.
Fig 5.
Effects of rhIL-26 on the activity and the quantity of proteinases in BAL samples.
Mice received intranasal instillation of recombinant human (rh) IL-26 protein, or its vehicle (PBS), with or without prior instillation of endotoxin (LPS). Bronchoalveolar lavage (BAL) samples were harvested 6 h (n = 8), 24 h (n = 10) and 72 h (n = 8) after instillations. The total activity of gelatinase (A) was measured with zymography in BAL samples that were pooled for each study group: the left panel presents the bands for MMP-9 and MMP-2 detected at all three time-points and the right panel shows the bands densitometry data of MMP-2 (upwards) and MMP-9 (downwards) (A). The respective net activity of (B) gelatinase and (F) elastase in the same BAL samples was measured utilizing a substrate-based method. The extracellular concentrations of gelatinases including (C) MMP-2, (D) MMP-9, (E) pro- MMP-9, and serine proteinases including (G) NE and (H) Cathepsin G (only in mice receiving instillation of LPS) were quantified in BAL samples using ELISA. Data are presented as mean.
Fig 6.
Effects of rhIL-26 on extracellular and intracellular proteinases in relation to innate effector cells in BAL samples.
Mice received prior intranasal instillation of endotoxin (LPS) with or without the subsequent addition of instillation of rhIL-26 protein or its vehicle (PBS) and bronchoalveolar lavage (BAL) samples were harvested. The average contents of extracellular proteinases per leukocyte in BAL samples were calculated to evaluate the activity of innate inflammatory cells. The intracellular contents of proteinases were measured using ELISA in whole cell lysates of BAL samples that were pooled for each study group. The panels show (A) MMP-2 per macrophage, (B) MMP-9 per leukocyte, (C) MPO per leukocyte, (D) NE per neutrophil, (E) intracellular MMP-2 per macrophage, (F) intracellular MMP-9 per leukocyte, (G) intracellular MPO per leukocyte and (H) intracellular NE per neutrophil. Data are presented as mean ± SEM.