Del-1 Plays a Protective Role against COPD Development by Inhibiting Inflammation and Apoptosis

Abstract

Neutrophilic inflammation is a prominent feature of chronic obstructive pulmonary disease (COPD). Developmental endothelial locus-1 (Del-1) has been reported to limit excessive neutrophilic inflammation by inhibiting neutrophil adhesion to the vascular endothelial cells. However, the effects of Del-1 in COPD are not known. We investigated the role of Del-1 in the pathogenesis of COPD. Del-1 protein expression was decreased in the lungs of COPD patients, especially in epithelial cells and alveolar macrophages. In contrast to human lung tissue, Del-1 expression was upregulated in lung tissue from mice treated with cigarette smoke extracts (CSE). Overexpression of Del-1 significantly suppressed IL-8 release and apoptosis in CSE-treated epithelial cells. In contrast, knockdown of Del-1 enhanced IL-8 release and apoptosis. In macrophages, overexpression of Del-1 significantly suppressed inflammatory cytokine release, and knockdown of Del-1 enhanced it. This anti-inflammatory effect was mediated by inhibiting the phosphorylation and acetylation of NF-κB p65. Nuclear factor erythroid 2-related factor 2 (Nrf2) activators, such as quercetin, resveratrol, and sulforaphane, increased Del-1 in both cell types. These results suggest that Del-1, mediated by Nrf2, plays a protective role against the pathogenesis of COPD, at least in part through anti-inflammatory and anti-apoptotic effects.

Keywords: COPD; Del-1; Nrf2; apoptosis; inflammation.

Figure 1

The expression level of Del-1 in human lung tissues. (A,B) Lung lysates from non-COPD (smokers without emphysema, n = 12) and COPD patients (smokers with emphysema, n = 12) were subjected to Western blot analysis for Del-1 and GAPDH. (B) Gel data were quantified using Scion image software Version 4.0. Data represent mean ± SE. ** p < 0.05. (C) Del-1 immunohistochemistry of lung tissues from non-COPD (smokers without emphysema, n = 6) and COPD patients (smokers with emphysema, n = 5). Arrows indicate the macrophages. Original magnifications, ×100 and ×200. HLT, human lung tissue; IHC, immunohistochemistry.

Figure 2

Intratracheal CSE instillation upregulated the expression levels of Del-1 mRNA and protein in lung tissues of C57BL/6 mice. (A–C) C57BL/6 mice were intratracheally instilled with saline or CSE, as described in the Materials and Methods section (saline n = 4, CSE n = 4). Mice were sacrificed at day 50 after the first instillation, to isolate lungs. (A) Total RNA was extracted, and quantitative real-time PCR for Del-1 and GAPDH was performed. Data represent mean ± SE. ** p < 0.05. (B) Lung lysates from mice were subjected to Western blot analysis for Del-1 and GAPDH. (C) Gel data were quantified using Scion image software Version 4.0. Data represent mean ± SE. ** p < 0.05. (D) Del-1 immunohistochemistry of lung tissue samples from saline- or CSE-treated mice. Arrows indicate the macrophages. Original magnifications, ×100 and ×200.

Figure 3

Del-1 suppressed CSE-induced IL-8 production and apoptosis in lung epithelial cells, without affecting aging marker expression or autophagy activation. HEK293T cells were transfected with pGIPZ−shCon, pGIPZ−shDel−1, Del1/pLOC, and pLOC, along with packaging vectors VSV−G and TRP, using lipofectamine reagent. A culture medium (1 mL) containing viral particles was added to BEAS-2B cells. Infected cells were used in experiments. (A) Total RNA was extracted, and quantitative real-time PCR for Del−1 and GAPDH was performed. Data represent mean ± SD. ** p < 0.05. (B) Cells were treated with CSE (1%) for 24 h. The concentration of IL-8 in cell supernatants was measured using ELISA. Data represent mean ± SD. ** p < 0.05. (C) Cells were treated with CSE (1%) for the indicated periods. Cell lysates were subjected to Western blotting for p21, LC3B, and GAPDH. (D) Cells were treated with CSE (2%) for 24 h. Cell viability was determined using LDH release assay. Data represent mean ± SD. ** p < 0.05. (E) Cell lysates were subjected to Western blot analysis for PARP, active caspase-3, and GAPDH. Results are representative of three separate experiments. LDH, lactose dehydrogenase; ELISA, enzyme-linked immunosorbent assay.

Figure 3

Del-1 suppressed CSE-induced IL-8 production and apoptosis in lung epithelial cells, without affecting aging marker expression or autophagy activation. HEK293T cells were transfected with pGIPZ−shCon, pGIPZ−shDel−1, Del1/pLOC, and pLOC, along with packaging vectors VSV−G and TRP, using lipofectamine reagent. A culture medium (1 mL) containing viral particles was added to BEAS-2B cells. Infected cells were used in experiments. (A) Total RNA was extracted, and quantitative real-time PCR for Del−1 and GAPDH was performed. Data represent mean ± SD. ** p < 0.05. (B) Cells were treated with CSE (1%) for 24 h. The concentration of IL-8 in cell supernatants was measured using ELISA. Data represent mean ± SD. ** p < 0.05. (C) Cells were treated with CSE (1%) for the indicated periods. Cell lysates were subjected to Western blotting for p21, LC3B, and GAPDH. (D) Cells were treated with CSE (2%) for 24 h. Cell viability was determined using LDH release assay. Data represent mean ± SD. ** p < 0.05. (E) Cell lysates were subjected to Western blot analysis for PARP, active caspase-3, and GAPDH. Results are representative of three separate experiments. LDH, lactose dehydrogenase; ELISA, enzyme-linked immunosorbent assay.

Figure 4

Del−1 suppressed LPS-induced pro-inflammatory chemokine/cytokines production in macrophages. (A–D) RAW264.7 cells were transiently transfected with control siRNA, Del−1 siRNA, pcDNA3.1 (control vector, C.V.), or Del-1 overexpression vector (Del−1 ov). After 48 h of transfection, cells were treated with LPS (100 ng/mL) for 6 h. (A,C) Total RNA was extracted and quantitative real-time PCR for Del-1 and GAPDH was performed. (B,D) The concentrations of KC, TNF−α, and IL−6 in cell supernatants were measured using ELISA. Data represent mean ± SD. ** p < 0.05. Results are representative of three separate experiments.

Figure 5

Del-1 did not affect the activation of MAP kinases. (A,B) RAW264.7 cells were transiently transfected with control siRNA, Del-1 siRNA, control vector (C.V.), or Del-1 overexpression vector (Del-1 ov). After 48 h of transfection, cells were treated with LPS (100 ng/mL) for the indicated times. Total cell extracts were subjected to Western blot analysis of phosphorylated p38 (p-p38), total p38 (p38), p-SAPK/JNK, total SAPK/JNK, p-ERK, and total ERK. Results are representative of three separate experiments.

Figure 6

Del−1 did not affect the degradation of IκBα or the nuclear translocation of NF−κB p65, but decreased levels of phospho−p65, NF−κB−DNA binding affinity, and NF-κB transcriptional activity. (A,B) RAW264.7 cells were transiently transfected with control siRNA, Del−1 siRNA, control vector (C.V.), or Del−1 overexpression vector (Del−1 ov). After 48 h of transfection, cells were treated with LPS (100 ng/mL) for the indicated times. Total cell extracts were subjected to Western blot analysis of IκB and GAPDH. (C–E) Transfected cells were treated with LPS for 1 h. Nuclear extracts were subjected to Western blot analysis of p65 and PARP (C). Total cell lysates were subjected to Western blot analysis of p−p65 (Ser536), acetyl−p65 (Lys310), total p65 (p65), and GAPDH (D,E). (F) RAW264.7 cells were transfected with C.V. or Del-1 overexpression vector. After 48 h of transfection, cells were treated with LPS (100 ng/mL) for 4 h. Nuclear proteins were extracted, and the DNA binding activity of p65 in nuclear extracts was measured. (G) Cells were transfected with NF−κB reporter plasmid, C.V., or Del−1 overexpression vector. After 48 h of transfection, cells were treated with LPS (100 ng/mL) for 19 h. Luciferase activity was determined. Data represent mean ± SD. ** p < 0.05. Results are representative of three separate experiments.

Figure 6

Del−1 did not affect the degradation of IκBα or the nuclear translocation of NF−κB p65, but decreased levels of phospho−p65, NF−κB−DNA binding affinity, and NF-κB transcriptional activity. (A,B) RAW264.7 cells were transiently transfected with control siRNA, Del−1 siRNA, control vector (C.V.), or Del−1 overexpression vector (Del−1 ov). After 48 h of transfection, cells were treated with LPS (100 ng/mL) for the indicated times. Total cell extracts were subjected to Western blot analysis of IκB and GAPDH. (C–E) Transfected cells were treated with LPS for 1 h. Nuclear extracts were subjected to Western blot analysis of p65 and PARP (C). Total cell lysates were subjected to Western blot analysis of p−p65 (Ser536), acetyl−p65 (Lys310), total p65 (p65), and GAPDH (D,E). (F) RAW264.7 cells were transfected with C.V. or Del-1 overexpression vector. After 48 h of transfection, cells were treated with LPS (100 ng/mL) for 4 h. Nuclear proteins were extracted, and the DNA binding activity of p65 in nuclear extracts was measured. (G) Cells were transfected with NF−κB reporter plasmid, C.V., or Del−1 overexpression vector. After 48 h of transfection, cells were treated with LPS (100 ng/mL) for 19 h. Luciferase activity was determined. Data represent mean ± SD. ** p < 0.05. Results are representative of three separate experiments.

Figure 7

Activators of Nrf2 increased Del-1 in lung epithelial cells and macrophages. BEAS-2B (A,B) and RAW264.7 cells (C,D) were transiently transfected with control siRNA or Nrf2 siRNA. After 48 h of transfection, cells were treated with quercetin, resveratrol, or sulforaphane (10 μM) for 24 h. Total RNA was extracted, and quantitative real-time PCR for Nrf2 and GAPDH was performed (A,C). Total cell lysates were subjected to Western blotting for Del-1 and GAPDH expression (B,D). Results are representative of three separate experiments. (E) The expression levels of Nrf2 in human lung tissues. Lung homogenates from non-COPD (smokers without emphysema, n = 11) and COPD patients (smokers with emphysema, n = 12) were subjected to Western blot analysis for Nrf2 and GAPDH. (F) Gel data were quantified using Scion image software Version 4.0. Data represent mean ± SE. ** p < 0.05.

Figure 7

Activators of Nrf2 increased Del-1 in lung epithelial cells and macrophages. BEAS-2B (A,B) and RAW264.7 cells (C,D) were transiently transfected with control siRNA or Nrf2 siRNA. After 48 h of transfection, cells were treated with quercetin, resveratrol, or sulforaphane (10 μM) for 24 h. Total RNA was extracted, and quantitative real-time PCR for Nrf2 and GAPDH was performed (A,C). Total cell lysates were subjected to Western blotting for Del-1 and GAPDH expression (B,D). Results are representative of three separate experiments. (E) The expression levels of Nrf2 in human lung tissues. Lung homogenates from non-COPD (smokers without emphysema, n = 11) and COPD patients (smokers with emphysema, n = 12) were subjected to Western blot analysis for Nrf2 and GAPDH. (F) Gel data were quantified using Scion image software Version 4.0. Data represent mean ± SE. ** p < 0.05.