Interleukin-10 down-regulates cathepsin B expression in fetal rat alveolar type II cells exposed to hyperoxia

Abstract

Purpose: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia.

Materials and methods: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR.

Results: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia.

Conclusion: Our study suggests that CB is enhanced in an oxygen- concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.

Fig. 1

Effect of hyperoxia on cytotoxicity, cathepsin B activity and IL-10 production. E19 type II cells exposed to 65%- and 85%-hyperoxia for the indicated periods of time. Cells in room air were used as controls. (A) Graphical depiction showing LDH-release expressed as experimental minus background LDH-release divided by maximum LDH-release in normoxia and 65%- and 85%-hyperoxic cells. The results are represented as mean±SD from 3 different experiments. (B) Graphical depiction showing activity of cathepsin B analyzed by fluorescence-based assay upon cell lysis. The results are represented as mean±SD from 3 different experiments. (C) Graphical depiction showing production of IL-10 in normoxia and 65%- and 85%-hyperoxic cells. The results are represented as mean±SD from 3 different experiments. LDH, lactate dehydrogenase; IL, interleukin; SD, standard deviation.

Fig. 2

Cytotoxicity of fetal type II cells after preincubation of cathepsin B-inhibitor or rIL-10. E19 type II cells exposed to 65%- and 85%-hyperoxia for the indicated periods of time. Cells in room air were used as controls. Graphical depiction showing LDH-release expressed as experimental minus background LDH-release divided by maximum LDH-release in cathepsin B-inhibitor- or rIL-10-untreated and treated cells. The results are represented as mean±SD from 3 different experiments. ^*p<0.01. CBI, cathepsin B-inhibitor; LDH, lactate dehydrogenase; rIL-10, recombinant IL-10; SD, standard deviation.

Fig. 3

Expression of cathepsin B in fetal type II cells exposed to hyperoxia. E19 type II cells exposed to 65%- and 85%-hyperoxia for the indicated periods of time. Cells in room air were used as controls. (A) Western blots showing that exposure to 65%- and 85%-hyperoxia for 12 h and 24 h increases the level of cathepsin B compared to the control samples. (B) Graphical depiction showing the relative intensities of cathepsin B from 3 independent experiments. Results are mean±SD from 3 different experiments. E19, embryonic day 19; SD, standard deviation.

Fig. 4

Expression of caspase-3 in fetal type II cells exposed to 65%- and 85%-hyperoxia. E19 type II cells exposed to 65%- and 85%-hyperoxia for the indicated periods of time. Cells in room air were used as controls. Western blots demonstrating that exposure of E19 cells to 65%- and 85%-hyperoxia for 12 h and 24 h does not increase the level of cleaved caspase-3 and does not concomitantly decrease the abundance of full-length procaspase-3 compared to control samples. Membranes were immunoblotted with anti-cleaved caspase-3 antibody, stripped, and reprobed with full-length procaspase-3 antibody. With actin antibody to control for protein loading. E19, embryonic day 19.

Fig. 5

IL-10 reduces cathepsin B expression in fetal type II cells exposed to hyperoxia. E19 type II cells exposed to 65%- and 85%-hyperoxia for the indicated periods of time. Cells in room air were used as controls. E19 cells were preincubated with 250 ng/mL rat recombinant IL-10 (rIL-10) before exposure to 65%- and 85%-hyperoxia. (A) Western blots showing that cathepsin B expression is reduced in rIL-10-treated cells compared to untreated cells. (B) Graphical depiction showing relative intensities of cathepsin B from 3 independent experiments in rIL-10-treated and untreated cells. (C) Graphical depiction showing gene expression of cathepsin B from 3 different experiments in rIL-10-treated and untreated cells. E19, embryonic day 19.