A Disintegrin and A Metalloproteinase-9 (ADAM9): A Novel Proteinase Culprit with Multifarious Contributions to COPD.

INTRODUCTION: Proteinases with a disintegrin and a metalloproteinase domain (ADAMs) have not been well studied in COPD. We investigated whether ADAM9 is linked to COPD in humans and mice. METHODS: ADAM9 blood and lung levels were measured in COPD patients versus controls, and air- versus cigarette smoke (CS)-exposed wild-type (WT) mice. WT and Adam9-/- mice were exposed to air or CS for 1-6 months, and COPD-like lung pathologies were measured. RESULTS: ADAM9 staining was increased in lung epithelial cells and macrophages in smokers and even more so in COPD patients and correlated directly with pack-year smoking history and inversely with airflow obstruction and/or FEV1 % predicted. Bronchial epithelial cell ADAM9 mRNA levels were higher in COPD patients than controls and correlated directly with pack-year smoking history. Plasma, BALF and sputum ADAM9 levels were similar in COPD patients and controls. CS exposure increased Adam9 levels in WT murine lungs. Adam9-/- mice were protected from emphysema development, small airway fibrosis, and airway mucus metaplasia. CS-exposed Adam9-/- mice had reduced lung macrophage counts, alveolar septal cell apoptosis, lung elastin degradation, and shedding of VEGFR2 and EGFR in BALF samples. Recombinant ADAM9 sheds EGF and VEGF receptors from epithelial cells to reduce activation of the Akt pro-survival pathway and increase cellular apoptosis. CONCLUSIONS: ADAM9 levels are increased in COPD lungs and linked to key clinical variables. Adam9 promotes emphysema development, and large and small airway disease in mice. Inhibition of ADAM9 could be a therapeutic approach for multiple COPD phenotypes.

The role of the CCR1 receptor in the inflammatory response to tobacco smoke in a mouse model.

OBJECTIVE: The aim was to create pathological changes in mice relevant to human smoke exposure that can be used to further understand the mechanisms and pathology of smoke-induced inflammatory disease. METHODS: Mice were exposed to tobacco smoke or lipopolysaccharide (LPS) to generate an inflammatory infiltrate within the lungs. RESULTS: Tobacco smoke exposure over a 4 day period led to neutrophilia in the lungs of BALB/c mice. Within the inflammatory exudates, significant changes were also seen in protein levels of IL-1B, IL-6, MIP-2, KC (IL-8) and TIMP-1 as measured by ELISA. Further protein changes, as measured via multiplex analysis revealed increased levels of MMP-9, MDC, LIF and MCP-1, amongst other mediators. Major changes in whole lung tissue gene expression patterns were observed. The neutrophilia seen after smoke exposure was steroid-insensitive, relative to doses of steroid needed to reduce LPS-driven neutrophilia in controls. This exposes pathological switches that are changed upon exposure to tobacco smoke, rendering steroids less effective under these conditions. Challenge of chemokine receptor type 1 (CCR1) KO mice in the tobacco smoke model showed that lack of this gene protected the mice from smoke-induced inflammation. CONCLUSIONS: This suggests the CCR1 receptor has a key role in the pathogenesis of smoke-induced inflammation.

TNF-alpha-induced self expression in human lung endothelial cells is inhibited by native and oxidized alpha1-antitrypsin.

Endothelial cells are among the main physiological targets of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In endothelial cells TNF-alpha elicits a broad spectrum of biological effects including differentiation, proliferation and apoptosis. alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases plays a vital role in protecting host tissue from proteolytic injury at sites of inflammation. Recently, it has been shown that AAT can be internalized by pulmonary endothelial cells, raising speculation that it may modulate endothelial cell function in addition to suppressing protease activity. Using Affymetrix microarray technology, real time PCR and ELISA methods we have investigated the effects of AAT on un-stimulated and TNF-alpha stimulated human primary lung microvascular endothelial cell gene expression and protein secretion. We find that AAT and TNF-alpha generally induced expression of distinct gene families with AAT exhibiting little activity in terms of inflammatory gene expression. Approximately 25% of genes up regulated by TNF-alpha were inhibited by co-administration of AAT including TNF-alpha-induced self expression. Surprisingly, the effects of AAT on TNF-alpha-induced self expression was inhibited equally well by oxidized AAT, a modified form of AAT, which lacks serine protease inhibitor activity. Overall, the pattern of gene expression regulated by native and oxidized AAT was similar with neither inducing pro-inflammatory gene expression. These findings suggest that inhibitory effects of native and oxidized forms of AAT on TNF-alpha stimulated gene expression may play an important role in limiting the uncontrolled endothelial cell activation and vascular injury in inflammatory disease.

Expression of genes involved in oxidative stress responses in airway epithelial cells of smokers with chronic obstructive pulmonary disease.

RATIONALE: The molecular mechanisms involved in airway oxidative stress responses reported in healthy smokers and in those with chronic obstructive pulmonary disease (COPD) are poorly understood. OBJECTIVES: To assess the expression of genes involved in oxidative stress responses in the bronchial epithelium of smokers with or without COPD and in relation to disease severity. METHODS: Global gene expression was assessed in bronchial brushings in 38 subjects with COPD, 14 healthy nonsmokers, and 18 healthy smokers. RESULTS: Gene expression analysis using Affymetrix arrays revealed mRNAs representing 341 out of 642 oxidative stress genes from two predefined gene sets to be differentially expressed in healthy nonsmokers when compared with healthy smokers, and 200 differentially expressed oxidative genes in subjects with COPD when compared with healthy smokers. Gene set enrichment analysis showed that pathways involved in oxidant/antioxidant responses were among the most differentially expressed gene pathways in smoking individuals, with further differences seen in COPD. Distinct, nonlinear gene expression patterns were identified across the severity spectrum of COPD, which correlated with the presence of certain transcription factor binding sites in their promoters. Significant changes in oxidant response genes observed in vivo were reproduced in vitro using primary bronchial epithelial cells from the same donors cultured at an air-liquid interface and exposed to cigarette smoke extract. CONCLUSIONS: Cigarette smoke induces significant changes in oxidant defense responses; some of these are further amplified, but not in a linear fashion, in individuals who develop COPD.

Inhibition of lipopolysaccharide-mediated human monocyte activation, in vitro, by alpha1-antitrypsin.

alpha1-Antitrypsin (AAT) is a major circulating and tissue inhibitor of serine proteinases. As such AAT is thought to play an important role in limiting host tissue injury at sites of inflammation. There is now increasing evidence, however, that AAT may exhibit biological activity independent of its protease inhibitor function. In this study we compared the effects of native (inhibitory) and modified (non-inhibitory), e.g., polymerised and oxidised forms of AAT on LPS-induced human monocyte activation, in vitro. We found that native AAT inhibited LPS-stimulated synthesis and release of TNFalpha and IL-1beta mRNA and protein, respectively, but enhanced the release of the anti-inflammatory cytokine, IL-10. Similarly, polymerised and oxidised forms of AAT inhibited LPS-stimulated IL-1beta and TNFalpha. The effects of AATs were observed whether added prior to or following removal of LPS, suggesting that sequestration of agonist was unlikely to explain their biological effects. Furthermore, studies with neutralising antibodies indicated that generation of IL-10 was unlikely to be the mechanism responsible for the inhibitory effects of AATs. Thus, our data demonstrate for the first time that AAT exhibits anti-inflammatory activity in vitro that is unrelated to inhibition of serine proteases.