Serum amyloid A in chronic obstructive pulmonary disease

Abstract

Introduction The Serum Amyloid A (SAA) protein family consists of differentially expressed apolipoproteins. There are four SAA genes responsible for encoding SAA in humans. SAA1 and SAA2 encode the acute phase SAA (A-SAA) which may increase by 1000 fold following an acute phase response (APR). SAA3 is a pseudogene and SAA4 encodes constitutive SAA (C-SAA) which is not augmented following an APR. Studies have shown SAA proteins to be biomarkers of disease activity as well contributors to the development and progression of disease. Production is primarily hepatic, while localised expression has been detected at sites of inflammation including the lungs. In patients with chronic obstructive pulmonary disease (COPO), serum A-SAA levels are higher than in healthy individuals, and rise further following an acute exacerbation of COPO (AECOPO). Inflammatory cells, cytokines as well as SAA are elevated in the lungs and the levels may reflect the severity of COPO and the predicted mortality. Smoking cessation, as well as current pharmacological regimes are not effective enough at reducing the lung inflammation in COPO, and therefore further understanding of the cytokine-mediated expression of SAA, might contribute towards the potential identification of novel therapeutic targets.

Aims This project aimed to study the cytokine-induced SAA transcriptional regulation in human airway-related cell line models. More specifically, it aimed to study the response of the SAA2 promoter to different concentrations of interleukin -1β (IL-β), interleukin-6 (IL-6), leukaemia inhibitory factor (LlF) and interleukin-8 (IL-8) in the alveolar epithelial A549 cell line and the monocytic-like human histiocytic lymphoma U937 cells. It also aimed to investigate temporal changes in serum SAA levels in stable COPO patients undergoing a 12- week pulmonary rehabilitation programme (PRP).

Methodology The cytokine-mediated activity of the SAA2 promoter in airway-related cells was investigated using reporter gene assays. A pGL4.1O-SAA2 luciferase reporter construct, was transfected into AS49 pulmonary epithelial cells and U937 monocytic cells using FuGENE® HD transfection reagent. HepG2 cells were also transfected as a positive control. Following 24 hours the cells were stimulated with different concentrations of IL-1β, IL-6, L1F and IL- 8. Six and 24 hours post-stimulation, SAA2 promoter activity was quantified using dual luciferase reporter assays. Stable (OPD patients from Mater Dei Hospital out patients' clinic, who were undergoing a PRP, as part of a separate research project, were recruited for SAA analysis. This programme was of 12-week duration, covering twice-weekly classes of 2 hours, including 1 hour of exercise and 1 hour of education. Respiratory parameters and blood samples were taken at week 0, week 8 and week 12. Serum SAA concentrations at these time-points were analysed using ELlSA. Results The SAA2 promoter has been shown to be cytokine-inducible in all cell lines used. Transcriptional upregulation of the SAA2 promoter in HepG2 cells (control) occurred with all cytokines. IL-1β induced the highest SAA2 transcriptional regulatory activity at 24 hours in U937 (27.8 fold over unstimulated cells) and HepG2 (10.1 fold) cells, at concentrations of 1.2ng/ml and 2ng/ml respectively. IL-8 at 40ng/ml induced a 5.7 fold increase in activity in U937 cells at 6 hours, while II 2.8 fold incrCu5C in SAA2 transcriptional activity was observed in AS49 with 20ng/ml of IL-8 at 24 hours. UF was only active in A549 cells, with maximal SAA promoter activity (3.0 fold) being observed at 40ng/ml of UF. IL-6 and L1F were ineffective in U937 cells. The median SAA concentration in stable (OPD patients was 38.1~g/ml with an interquartile range (IQR) of 52.0µg/ml at baseline, 61.6µg/ml (IQR of 85.4µg/ml) after 8 weeks (p<0.05 compared to baseline), and 41.4µg/ml (IQR of 74.3µg/ml) at week 12.

Conclusion Inflammatory microenvironments can induce SAA2 transcription in airway-related cells, with the promoter being most active in IL-β-stimulated monocytes. This may have potential implications in understanding the interrelationships between these components of the pro-inflammatory network, and interpretation of their levels as clinical biomarkers. The observed PRP-related serum SAA level changes in COPD patients require further study in order to better understand the underlying mechanisms.