Non-invasive assessment of small airway remodelling in smokers.

Smoking associated COPD progression is likely to be directly linked to differential injury and repair dynamics in small airways (SA). Although IL8 is a well-accepted marker for injured airway epithelium, Clara cells [the predominant proliferating cells in SA] and SCGB1A1 protein [their major secretory product] have only recently emerged as potential SA repair markers. We therefore postulate that the SCGB1A1/IL8 ratio in the airways of smokers would be inversely associated with physiological, radiological and clinical measures of COPD. A cross-sectional cohort of 28 smokers undergoing surgery for peripheral nodule was recruited (24M/4F, age 61 +/- 11 y, FEV1s 76 +/- 20%, smoking 40 +/- 12 p.y). SCGB1A1 and IL8 were measured by ELISA in the induced sputum (IS) 3 to 5 days prior to surgery as well as by immunohistochemistry from lung tissue (also assessed morphometrically) obtained distant to the cancer surgery site. COPD was assessed using standard clinical, functional and radiological parameters. Log-transformed IS-SCGB1A1 was linearly correlated with SCGB1A1-positive epithelial cells detected via immunohistochemistry (r = .533, p = .001), while IS-IL8 was positively related to SA infiltrating neutrophils (Elastase-positive cells). There was a striking negative correlation between IS-SCGB1A1/IL8 levels and whole airway thickness [SA < 2 mm] at morphometry (r = -0.83, p < 0.0001). IS-SCGB1A1/IL8 levels were also inversely associated with nitrogen slope [r = -0.52, p < 0.001] and HRCT SA score [r = -0.51, p < 0.001]. In a multivariate analysis the IS-SCGB1A1/IL8 ratio was a stronger predictor than both the physiological and radiological measures of SA disease assessed. The SCGB1A1/IL8 ratio measured in sputum is a potentially valuable biomarker for non-invasive assessment of SA remodelling in smokers.

Synthesis and anti-inflammatory effect of lipoxins in human airway epithelial cells.

In this study, we investigated the synthesis of lipoxins (LXs) and their anti-inflammatory effects in different human airway epithelial cell culture models. After cell incubation with exogenous 5(S),6(R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, LXA(4) was detected in supernatants of differentiated human bronchial epithelial cells by contrast to non-differentiated cells. Exogenous LXA(4) significantly inhibited tumor necrosis factor-alpha (TNF-alpha)-induced interleukin-8 (IL-8) release in the different epithelial cell types and the potency of inhibition was dependent of the accessibility of the specific LXA(4) receptor, formyl-peptide receptor like-1 (FPRL-1) expressed by all these cells. Immunohistochemistry analysis on human bronchial biopsies showed a high expression of FPRL-1 in the epithelium. Finally, an FPRL-1 receptor antagonist, boc-2 peptide reversed LXA(4) effect on IL-8 generation. Together, these findings indicate that differentiated human bronchial epithelium synthesizes LX in vivo which could have autocrine actions through its specific receptor FPRL-1 to promote resolution of airway inflammation.

Lipoxin A4 stimulates a cytosolic Ca2+ increase in human bronchial epithelium.

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells.

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.