Neutrophil-Derived Elastase Induces TGF-β1 Secretion in Human Airway Smooth Muscle via NF-κB Pathway

Abstract

Neutrophils are infiltrated in airways of individuals with more severe and chronic asthma, with uncertain significance. Airway smooth muscle (ASM), apart from its contractile properties, is critically involved in the pathogenesis of asthma by producing inflammatory mediators. In the present study, we investigated the impact of neutrophil-derived elastase (NE) on ASM in terms of TGF-beta1 release, and we explored the underlying mechanisms. Primary ASM cells were serum starved for 24 h before stimulation with NE (0.01-0.5 microg/ml). TGF-beta1 in supernatant was determined by ELISA and mRNA quantified by real-time RT-QPCR. NF-kappaB nuclear translocation and activation was examined by Western blotting and kappaB-2 dEGFP reporter gene assay. Association of IL-1 receptor-associated kinase (IRAK) with MyD88 was studied by co-immunoprecipitation and Toll-like receptor 4 (TLR4) determined by FACS scan and Western blotting. We demonstrated that NE enhanced TGF-beta1 release in a time-dependent manner. This induction was inhibited by actinomycin D (5 mM), cycloheximide (5 mM), and NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC, 1 mM), aspirin (2.5 mM), and sodium salyicylate (2.5 mM). Stimulation with NE was rapidly followed by association of IRAK with MyD88, phosphorylation of IkappaBalpha, and nuclear translocation of p65 with increased transactivation activity. We also found that TLR4 levels were reduced upon NE treatment. These data suggest that NE upregulates TGF-beta1 gene expression and release via My88/IRAK/NF-kappaB pathway, possibly through activation of TLR4, and shed light on a potential role of neutrophils in the pathogenesis of asthma.

Neutrophil Elastase Represses IL-8/CXCL8 Synthesis in Human Airway Smooth Muscle Cells through Induction of NF-κB Repressing Factor

“…In contrast to the comprehensive understanding of the NE-induced biological effect on respiratory epithelium, little is known about that on ASM. Previously, we have reported that NE also stimulates the TLR4/MyD88/TRAF6/NF-B pathway in primary human ASM (hASM) cells, leading to induction of TGF-␤1 transcription (17). However, despite NE activation of NF-B in hASM cells, NE was found to suppress the basal secretion of IL-8/CXCL8 in our preliminary study.…”

“…In addition to its direct antibacterial effects, increasing evidence indicates that NE can provoke a variety of cellular responses in respiratory epithelial cells. By activating specific intracellular signal pathways, including NF-B, MAPK, and protein kinase C (13)(14)(15)(16)(17), NE induces expression of a number of genes, such as IL-8/CXCL8 (13,14,18), cathepsin B, matrix metalloprotease-2 (15), and MUC5AC (19). NE has been shown to activate NF-B through the TLR4/MyD88/ TRAF6 pathway, leading to induction of IL-8/CXCL8 expression in bronchial and lung epithelial cells (14,20,21).…”

Airway Inflammation after Bronchial Thermoplasty for Severe Asthma

“…One of these, TGF-b, has a complex role in the pathogenesis of severe asthma (35). It is produced by several cell types, including epithelial cells, eosinophils, macrophages, fibroblasts, and helper T cells (36,37), and is involved in epithelial transformation, subepithelial fibrosis, airway smooth muscle remodeling, microvascular changes, mucus production, and both suppressing and activating inflammatory cytokines (38)(39)(40)(41). TGF-b 1 is a major regulator as well as effector in the immune response.…”

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

“…36 A similar mechanism of action may function in DCs, since we presently report that elastase and even PMN CS decrease IB␣ protein expression. The fact that pretreatment with SLPI prevented the PMN CS-induced IB␣ degradation further supports the role of elastase.…”

“…41 Interestingly, elastase has been shown to increase the release of TGF-␤1 by airway smooth muscle. 36 Furthermore, lung DCs have an immature phenotype. 42 Recently, Roghanian et al 43 showed that sputum from chronic obstructive pulmonary disease and patients with cystic fibrosis or elastase down-regulated the expression of CD40, CD80, CD86 (but not MHCII) on murine DCs, inhibited LPSinduced murine DC maturation and T-cell proliferation.…”