Sabatini et al

Sabatini et al., (2010) have shown that PDE4 inhibition was dependent on the presence of PGE2 [45]. or 4% in combination with LPS at 0.1 g/ml. After 24 hours cell culture supernatants were collected and chemokines were quantified by ELISA. Results are expressed as means SEM of 3 impartial experiments. * p<0.05 compared to control; # p<0.05 compared to vehicle.(TIF) pone.0085243.s002.tif (439K) GUID:?AF7F1838-21D0-40C0-A9D5-002644BFB02F Abstract Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 g/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5C120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E2 alone Gemilukast or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of Gemilukast Gemilukast LPS 0.1 g/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE2 reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT TEF2 pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is usually reduced by RNO. Introduction Chronic obstructive pulmonary disease (COPD) is usually a major and a growing cause of morbidity and mortality worldwide. COPD is usually characterized by airflow limitation that is not fully reversible [1]. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs [2]. The major triggering factor is usually cigarette smoking, which accounts for 80C90% of Gemilukast the COPD cases. The cigarette smoke causes airway inflammation by activating epithelial cells and macrophages, which by releasing proteases and free oxygen radicals cause injury of parenchyma tissue. These cells can also release inflammatory mediators, including cytokines and chemokines such as IL-8/CXCL8, monocyte chemotactic peptide-1 (MCP-1/CCL2) and Growth-related oncoprotein- (Gro-/CXCL1). These chemokines play a role in mechanisms leading to inflammatory process in airways and progressive airflow limitation [3], [4], [5]. Besides, there is now increasing amount of evidence that chronic colonization of airways by respiratory pathogens, predominantly gram-negative bacteria, contributes to the progression of COPD and is also responsible for the persistent airway inflammation [6], [7]. Several signaling pathways, such as mitogen activated protein kinase (MAPK) control the expression of these chemokines as exhibited by taking advantage of selective inhibitors or siRNA strategies [8], [9], [10], [11]. Indeed, inhibitors of ERK 1/2 and p38 MAP kinases the decreased the release of cytokines induced by cigarette Gemilukast smoke in airway epithelial cells [12], [13]. Other protein kinases may be involved in inflammatory responses like Scr family kinases, JAKs (Janus kinases) as well as their downstream transcription factors of the STAT (signal transducer and activator of transcription) family [14], [15]. Phosphodiesterase 4 (PDE4) inhibitors, in view of their antiinflammatory effects, have recently been confirmed as a.