Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy).

PURPOSE: There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. METHODS: THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. RESULTS: We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. CONCLUSIONS: The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.

The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

Chronic inflammatory diseases of the airways are associated with gastro-oesophageal reflux (GOR) and aspiration events. The observation of lipid-laden macrophages (LLMs) within the airway may indicate aspiration secondary to GOR. The proposed mechanism, that lipid droplets from undigested or partially digested food are aspirated leading to accumulation in scavenging macrophages, led us to hypothesise that an activated population of LLMs could interact with other immune cells to induce bronchial inflammation. To test this, we generated an in vitro model using differentiated THP-1 cells, which were treated with a high-fat liquid feed. Here, we show that THP-1 cells can take up lipid from the high-fat feed independent of actin polymerisation or CD36-dependent phagocytosis. These cells did not exhibit M1 or M2 polarisation. Gene array analysis confirmed over 8000 genes were upregulated by at least twofold following high fat exposure, and IL-8 was the most upregulated gene. Pathway analysis revealed upregulation of genes known to be involved in chronic obstructive pulmonary disease (COPD) pathophysiology. We suggest that aspiration and macrophage phagocytosis may be important mechanisms in the aetiology of diseases such as COPD and cystic fibrosis that are characterised by high levels of IL-8 within the airways.

Continuous Cough Monitoring Using Ambient Sound Recording During Convalescence from a COPD Exacerbation.

PURPOSE: Cough is common in chronic obstructive pulmonary disease (COPD) and is associated with frequent exacerbations and increased mortality. Cough increases during acute exacerbations (AE-COPD), representing a possible metric of clinical deterioration. Conventional cough monitors accurately report cough counts over short time periods. We describe a novel monitoring system which we used to record cough continuously for up to 45 days during AE-COPD convalescence. METHODS: This is a longitudinal, observational study of cough monitoring in AE-COPD patients discharged from a single teaching hospital. Ambient sound was recorded from two sites in the domestic environment and analysed using novel cough classifier software. For comparison, the validated hybrid HACC/LCM cough monitoring system was used on days 1, 5, 20 and 45. Patients were asked to record symptoms daily using diaries. RESULTS: Cough monitoring data were available for 16 subjects with a total of 568 monitored days. Daily cough count fell significantly from mean ± SEM 272.7 ± 54.5 on day 1 to 110.9 ± 26.3 on day 9 (p < 0.01) before plateauing. The absolute cough count detected by the continuous monitoring system was significantly lower than detected by the hybrid HACC/LCM system but normalised counts strongly correlated (r = 0.88, p < 0.01) demonstrating an ability to detect trends. Objective cough count and subjective cough scores modestly correlated (r = 0.46). CONCLUSIONS: Cough frequency declines significantly following AE-COPD and the reducing trend can be detected using continuous ambient sound recording and novel cough classifier software. Objective measurement of cough frequency has the potential to enhance our ability to monitor the clinical state in patients with COPD.

Objective Measurement of Cough Frequency During COPD Exacerbation Convalescence.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. Cough and sputum production are associated with adverse outcomes in COPD and are common during COPD exacerbation (AE-COPD). This study of objective cough monitoring using the Hull Automated Cough Counter and Leicester Cough Monitor software confirms that this system has the ability to detect a significant decrease in cough frequency during AE-COPD convalescence. The ability to detect clinically meaningful change indicates a potential role in home monitoring of COPD patients.

Increased inducible heat shock protein 72 expression associated with PBMC isolated from patients with haematological tumours.

BACKGROUND: Heat shock protein 72 (Hsp72) is a highly inducible stress protein and molecular chaperone. Cancers have been shown to be associated with increased Hsp72 expression within the tumour itself and this may lead to resistance to apoptosis. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from patients diagnosed with chronic lymphocytic leukaemia (CLL) (n = 27) and chronic myelomonocytic leukaemia (CMML) (n = 16) and Hsp72 expression was characterized on both the cell surface and intracellularly by flow cytometry. To allow for comparison PBMC from breast cancer patients (n = 25) and healthy volunteers (n = 19) were included. RESULTS: Both lymphocytes and monocytes from CLL and CMML patients showed high levels of total Hsp72 expression (4-6 fold increase) in comparison to breast cancer and healthy subjects. The majority of Hsp72 in these tumours was determined to be cell-surface expressed (64-93% of cell total Hsp72). CONCLUSIONS: A correlation was observed between lymphocyte and monocyte total Hsp72 expression (p < 0.001) suggesting a common stress response pathway may exist in these blood cells and there are stress conditions present within the circulation. Hsp72 expression was not found to be related to white blood cell count.