ABSTRACT
Title
A possible involvment of Monocyte-derived MicroParticles (MdMP) in alveolar macrophages inflammatory response
Authors
A Amoruso1, P Balbo2, C Bardelli1, D Federici-Canova1, LG Fresu1 & S Brunelleschi1
1Dept. of Medical Sciences, University “A. Avogadro”, Novara (Italy);
2Dept. of Respiratory Medicine, Azienda Ospedaliera Maggiore della Carità, Novara (Italy);
1Dept. of Medical Sciences, University “A. Avogadro”, Novara (Italy);
2Dept. of Respiratory Medicine, Azienda Ospedaliera Maggiore della Carità, Novara (Italy);
Abstract
Microparticles (MP), small membrane-bound particles originating from different cell types during activation or apoptosis, mediate intercellular communications, exert pro-coagulant activity and affect inflammation and other pathophysiological conditions (1-3). Peroxisome Proliferator-Activated Receptor-gamma (PPARγ ) might represent a novel target in lung diseases (4), largely due to its ability to down-regulate inflammatory mediators, and rosiglitazone has been shown to improve lung function in smokers with asthma (5). Moreover, a deficiency in PPARγ activity and a concomitant NF-κB activation was evidenced in alveolar macrophages (AM) from patients with sarcoidosis (6).
We sought to comprehensively characterize the effects of Monocyte-derived MPs (MdMP) on the PPARγ -dependent anti-inflammatory pathway in human alveolar macrophages and in peripheral healthy-donor monocyte/macrophages. MdMP have been little investigated and, to our knowledge, never evaluated for their possible autologous effects.
MdMP were obtained from supernatants of cultured human monocytes stimulated by the calcium ionophore A23187 (12 μM), and characterized. AM were isolated from Broncho-Alveolar Lavage (BAL, mainly performed for diagnostic purpose) of 11 patients with sarcoidosis and 8 patients with other lung diseases (7): the fluid obtained was filtered and centrifuged (400 x g, 30 min). The whole BAL pellet was washed twice in PBS, resuspended in RPMI 1640 medium supplemented with 10 % foetal bovine serum, 2 mM glutamine and antibiotics, and plated (2 h, 37°C, 5% CO2). Non-adherent cells (mainly lymphocytes) were gently removed and AM used for the experiments; differential cell count, phenotypical analysis and CD4+/CD8+ ratio were evaluated. Cells were challenged by MdMP or phorbol-12-myristate 13-acetate (PMA, used as standard stimulus), in the absence or presence of PPARγ agonists and antagonists. Superoxide anion production (measured spectrophotometrically), cytokine release (ELISA kits), PPARγ protein expression (immunoblotting) and NF-κB activation (EMSA assay) were evaluated.
Our results demonstated that MdMP induce, in a concentration-dependent manner, oxy-radical production, cytokine release and NF-κB activation in AM as in human monocytes/macrophages. In all cell types, MP-induced effects are inhibited by PPARγ agonists (rosiglitazone and 5-deoxy-D12,14-prostaglandin J2) and reversed by a PPARγ antagonist (GW9662). AM from sarcoidosis patients present a constitutive PPARγ expression similar to monocytes and significantly lower compared to AM from patients with other lung diseases. The latter population instead presents a similar level of this receptor to monocytes-derived macrophages. This study proves that, in human AM, monocyte-derived MP exert important pro-inflammatory effects, (increasing superoxide anion production, cytokine release and NF-κB activation) that are reduced by PPARγ agonists, but can also induce anti-inflammatory response (increasing PPARγ expression). Interestingly, this dual role of MdMP on monocytes and AM populations, can suggest a mechanism of auto-stimulation in the monocyte/macrophage lineage.
(1) Nomura S et al (2008) Thromb Res; 123: 8-23.
(2) Anderson HC et al (2010) Lab Invest; 90:1549-1557.
(3) Hugel B et al (2005) Physiology; 20: 22-27.
(4) Belvisi Mg et al (2009). Br J Pharmacol; 158: 994-1003.
(5) Spears M et al (2009) Clin Pharmacol Ther; 86: 49-53.
(6) Culver DA et al (2004) Am J Respir Cell Mol Biol; 30: 1-5.
(7) Bardelli C.et al (2005) Br J Pharmacol; 145:385-396.
(8) Brunelleschi S et al (1996) Neuropeptides; 30: 456-464.
Acknowledgements. This work was supported by PRIN 20074S9KXF grants.
We sought to comprehensively characterize the effects of Monocyte-derived MPs (MdMP) on the PPARγ -dependent anti-inflammatory pathway in human alveolar macrophages and in peripheral healthy-donor monocyte/macrophages. MdMP have been little investigated and, to our knowledge, never evaluated for their possible autologous effects.
MdMP were obtained from supernatants of cultured human monocytes stimulated by the calcium ionophore A23187 (12 μM), and characterized. AM were isolated from Broncho-Alveolar Lavage (BAL, mainly performed for diagnostic purpose) of 11 patients with sarcoidosis and 8 patients with other lung diseases (7): the fluid obtained was filtered and centrifuged (400 x g, 30 min). The whole BAL pellet was washed twice in PBS, resuspended in RPMI 1640 medium supplemented with 10 % foetal bovine serum, 2 mM glutamine and antibiotics, and plated (2 h, 37°C, 5% CO2). Non-adherent cells (mainly lymphocytes) were gently removed and AM used for the experiments; differential cell count, phenotypical analysis and CD4+/CD8+ ratio were evaluated. Cells were challenged by MdMP or phorbol-12-myristate 13-acetate (PMA, used as standard stimulus), in the absence or presence of PPARγ agonists and antagonists. Superoxide anion production (measured spectrophotometrically), cytokine release (ELISA kits), PPARγ protein expression (immunoblotting) and NF-κB activation (EMSA assay) were evaluated.
Our results demonstated that MdMP induce, in a concentration-dependent manner, oxy-radical production, cytokine release and NF-κB activation in AM as in human monocytes/macrophages. In all cell types, MP-induced effects are inhibited by PPARγ agonists (rosiglitazone and 5-deoxy-D12,14-prostaglandin J2) and reversed by a PPARγ antagonist (GW9662). AM from sarcoidosis patients present a constitutive PPARγ expression similar to monocytes and significantly lower compared to AM from patients with other lung diseases. The latter population instead presents a similar level of this receptor to monocytes-derived macrophages. This study proves that, in human AM, monocyte-derived MP exert important pro-inflammatory effects, (increasing superoxide anion production, cytokine release and NF-κB activation) that are reduced by PPARγ agonists, but can also induce anti-inflammatory response (increasing PPARγ expression). Interestingly, this dual role of MdMP on monocytes and AM populations, can suggest a mechanism of auto-stimulation in the monocyte/macrophage lineage.
(1) Nomura S et al (2008) Thromb Res; 123: 8-23.
(2) Anderson HC et al (2010) Lab Invest; 90:1549-1557.
(3) Hugel B et al (2005) Physiology; 20: 22-27.
(4) Belvisi Mg et al (2009). Br J Pharmacol; 158: 994-1003.
(5) Spears M et al (2009) Clin Pharmacol Ther; 86: 49-53.
(6) Culver DA et al (2004) Am J Respir Cell Mol Biol; 30: 1-5.
(7) Bardelli C.et al (2005) Br J Pharmacol; 145:385-396.
(8) Brunelleschi S et al (1996) Neuropeptides; 30: 456-464.
Acknowledgements. This work was supported by PRIN 20074S9KXF grants.