Concentration-dependent effects of native and polymerised α1-antitrypsin on primary human monocytes, in vitro

BACKGROUND: α1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non-inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-κB. RESULTS: We found that native and polymerised AAT at lower concentrations, such as 0.1 mg/ml, enhance expression of TNFα (10.9- and 4.8-fold, p < 0.001), IL-6 (22.8- and 23.4-fold, p < 0.001), IL-8 (2.4- and 5.5-fold, p < 0.001) and MCP-1 (8.3- and 7.7-fold, p < 0.001), respectively, compared to buffer exposed cells or cells treated with higher doses of AAT (0.5 and 1 mg/ml). In parallel to increased cytokine levels, low concentrations of either conformation of AAT (0.02–0.1 mg/ml) induced NF-κB p50 activation, while 1 mg/ml of either conformation of AAT suppressed the activity of NF-κB, compared to controls. CONCLUSIONS: The observations reported here provide further support for a central role of AAT in inflammation, both as a regulator of proteinase activity, and as a signalling molecule for the expression of pro-inflammatory molecules. This latter role is dependent on the concentration of AAT, rather than on its proteinase inhibitory activity

Circulating monocytes from healthy individuals and COPD patients

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by incompletely reversible airflow obstruction associated with inflammation in which monocytes/macrophages are the predominant inflammatory cells. The only known genetic factor related to COPD is inherited PiZZ deficiency of α1-antitrypsin (AAT), an inhibitor of serine proteases. METHODS: We investigated the basal and LPS-stimulated release of pro-inflammatory molecules from blood monocytes isolated from age and gender matched healthy (n = 30) and COPD (n = 20) individuals with and without AAT deficiency. RESULTS: After 18 h of cell culture the basal release of MMP-9 was 2.5-fold, p < 0.02 greater, whereas IL-8 was 1.8-fold (p < 0.01) lower from COPD patient monocytes than from controls. LPS-stimulated release of IL-6 and MCP-1 was greater from COPD patient's monocytes relative to controls, while activation of control cells resulted in enhanced secretion of ICAM-1 and MMP-9 compared to COPD patients. Independent of disease status, monocytes from PiZZ AAT carriers released less TNFα (by 2.3-fold, p < 0.03). CONCLUSIONS: The basal and LPS-stimulated secretion of specific pro-inflammatory molecules from circulating monocytes differs between healthy and COPD subjects. These findings may be valuable for further studies on the mechanisms involved in recruitment and activation of inflammatory cells in COPD

Chronic Obstructive Pulmonary Disease: Role of Alpha-1-antitrypsin

The only proven genetic risk factor for Chronic Obstructive Pulmonary Disease (COPD) is an inherited Z (Glu342¡úLys) deficiency of alpha-1-antitrypsin (AAT), a major inhibitor of neutrophil elastase. In vivo, AAT is found in both native (inhibitory) and modified (non-inhibitory) forms. There is now increasing evidence that the different forms of AAT may exhibit biological activities independent of protease inhibition. The aim of my studies was to validate polymerized (non inhibitory) form of AAT as a potential COPD biomarker and to investigate its biological effects in vitro. We have discovered that a mouse monoclonal antibody, ATZ11, raised against the mutant Z AAT, does not detect the mutation per se, but recognizes a conformation-dependent epitope in polymerized and elastase-complexed AAT. By using this antibody we show that in Z deficiency subjects a predominant fraction of plasma AAT is in a polymerized form. In addition, a positive staining of endothelial cells with ATZ11 is detected in both wild-type M and deficiency Z individuals. The levels of total and polymerized serum AAT and inflammatory biomarkers in M and Z COPD patients and controls were correlated. Hypothetically, by using factor analysis, we were able to segregate the variables measured into two independent components: the first containing MMP9, MCP-1, IL-8 and VEGF and the second ¨C total and polymerized AAT, sE-selectin and ICAM-1. We demonstrate that 95% of originally grouped individuals can be correctly classified on the basis of the measured variables. This suggests that the combinations of biomarkers may provide useful diagnostic tools. We next investigated the release of pro-inflammatory molecules by monocytes isolated from blood of COPD patients and controls with and without Z AAT deficiency under basal conditions and after stimulation with endotoxin. Dependent on disease state and AAT genotype the different profiles of pro-inflammatory molecules are released by monocytes. The development of COPD may also be ascribed to acquired AAT deficiency, which results from the post-translational modifications of the protein. We have tested whether native and modified (polymerized) forms of AAT differ in their effects on primary human monocytes in vitro. Both native and polymerized AAT exhibit similar pro-inflammatory effects at lower, but not at physiological, concentrations. These properties of AAT appear to be dependent on protein concentration, but not on molecular form. Our studies support a central role of AAT in inflammation and serve as a basis for the future research in identifying new biological role(s) of AAT in vivo