Circulating monocytes from healthy individuals and COPD patients

Abstract

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 alpha1-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 TNFalpha (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.

Figure 1

LPS-stimulated release of pro-inflammatory molecule from COPD patient monocytes relative to controls. Isolated blood monocytes from COPD patients (n = 20) and controls (n = 30) were activated with LPS (1 μg/ml) for 18 h. Pro-inflammatory molecular release was measured by ELISA methods. Monocytes from patients with COPD released greater levels of IL-6 and MCP-1, relative to controls (A), while the activation of control cells resulted in an enhanced secretion of ICAM-1 and MMP-9 (B).

Figure 2

Monitoring of NFκB activation in monocytes from patients with COPD and controls by ELISA-based TransAM kit^AMCell nuclear extracts were prepared from monocytes isolated from COPD patients and controls with and without PiZZ AAT deficiency. NFκB activity assay was performed in a 96-well plates with 10 μg of cell extract per well. Absorbance was measured by spectrophotometry at 405 nm. A positive control performed by using a nuclear extract derived from Jurkat cells. This extract is optimized to give a strong signal when used at 2.5 μg/well. Specificity controls were performed by adding a molar excess (20 pmol/well] of mutant NFκB oligonucleotide [the positive signal remained unaffected] and wild-type NFκB oligonucleotide (a signal was abolished). The bars represent mean values of three independent measurements ± SD.