Alessandro Celi

Monocyte/Macrophage-Derived Microparticles Up-Regulate Inflammatory Mediator Synthesis by Human Airway Epithelial Cells

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

Role of NF-κB and PPAR-γ in lung inflammation induced by monocyte-derived microparticles

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-&kgr;B transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-&kgr;B activation and is modulated by PPAR-&ggr;. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-&kgr;B translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-&kgr;B activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-&ggr; agonists, rosiglitazone and 15-deoxy-&Dgr;12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-&ggr; agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-&kgr;B activation through a PPAR-&ggr; dependent pathway.

12-Hydroxyeicosatetraenoic acid upregulates P-selectin-induced tissue factor activity on monocytes

12‐Hydroxyeicosatetraenoic acid (12‐HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P‐selectin‐stimulated monocytes in a time‐ and dose‐dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P‐selectin cDNA. Both 5‐HETE and leukotriene C4 are inactive in this system. Furthermore, the effect is not dependent on non‐specific monocyte adhesion, since monocytes incubated with CHO cells expressing E‐selectin do not express tissue factor, either in the presence or in the absence of 12‐HETE. These results show that 12‐HETE is a cofactor for the expression of tissue factor by monocytes.

Procoagulant, Tissue Factor-Bearing Microparticles in Bronchoalveolar Lavage of Interstitial Lung Disease Patients: An Observational Study

Coagulation factor Xa appears involved in the pathogenesis of pulmonary fibrosis. Through its interaction with protease activated receptor-1, this protease signals myofibroblast differentiation in lung fibroblasts. Although fibrogenic stimuli induce factor X synthesis by alveolar cells, the mechanisms of local posttranslational factor X activation are not fully understood. Cell-derived microparticles are submicron vesicles involved in different physiological processes, including blood coagulation; they potentially activate factor X due to the exposure on their outer membrane of both phosphatidylserine and tissue factor. We postulated a role for procoagulant microparticles in the pathogenesis of interstitial lung diseases. Nineteen patients with interstitial lung diseases and 11 controls were studied. All subjects underwent bronchoalveolar lavage; interstitial lung disease patients also underwent pulmonary function tests and high resolution CT scan. Microparticles were enumerated in the bronchoalveolar lavage fluid with a solid-phase assay based on thrombin generation. Microparticles were also tested for tissue factor activity. In vitro shedding of microparticles upon incubation with H2O2 was assessed in the human alveolar cell line, A549 and in normal bronchial epithelial cells. Tissue factor synthesis was quantitated by real-time PCR. Total microparticle number and microparticle-associated tissue factor activity were increased in interstitial lung disease patients compared to controls (84±8 vs. 39±3 nM phosphatidylserine; 293±37 vs. 105±21 arbitrary units of tissue factor activity; mean±SEM; p<.05 for both comparisons). Microparticle-bound tissue factor activity was inversely correlated with lung function as assessed by both diffusion capacity and forced vital capacity (r2 = .27 and .31, respectively; p<.05 for both correlations). Exposure of lung epithelial cells to H2O2 caused an increase in microparticle-bound tissue factor without affecting tissue factor mRNA. Procoagulant microparticles are increased in interstitial lung diseases and correlate with functional impairment. These structures might contribute to the activation of factor X and to the factor Xa-mediated fibrotic response in lung injury.

Presence and persistence of serum anti-benzo[apyrene diolepoxide-DNA adduct antibodies in smokers: Effects of smoking reduction and cessation

Among biomarkers of tobacco smoke (TS)‐induced genotoxic damage, benzo[a]pyrene diolepoxide‐DNA adducts (BPDE‐DNA) are extensively studied. Adducted DNA becomes antigenic and antibodies anti‐BPDE‐DNA (BPDE‐DNA‐Abs) may be found in serum of exposed subjects. Little is known about the persistence of BPDE‐DNA, and no study has been performed to evaluate the persistence of BPDE‐DNA‐Abs after cessation of exposure. Fifty heavy smokers, enrolled in a smoking cessation program with nicotine patch substitution therapy, were evaluated for the presence of BPDE‐DNA‐Abs before (w0) and 1, 3, 6 and 12 weeks (w1–12) after the start of the program. Nicotine or placebo patches were randomly assigned to the subjects. BPDE‐DNA‐Abs were determined in serum by non‐competitive ELISA. After the start of the cessation program, 28 subjects quit smoking (group Q) and the other 22 reduced by about 75% the number of cigarettes smoked per day (group R). At the start of the program (w0) 8% of subjects were positive. At w1 the prevalence of positivity had increased both in subjects who quit smoking (Q: 21%) and in subjects who had reduced the number of cigarettes per day (R: 27%). Positivity remained stable up to w12 (21%) for group Q, whereas it increased to 41% in group R. Serum BPDE‐DNA‐Abs can be detected in smokers, and their persistence for months after smoking cessation suggests their usefulness for relatively long‐term surveys. The low percentage of positivity in actual heavy smokers and the increase in antibody positivity with smoking cessation or reduction must be taken into account when interpretating serum BPDE‐DNA‐Ab measurement in exposed individuals. Int. J. Cancer, 70:145–149, 1997.

Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization

ObjectivesMicroparticles are membrane vesicles shed by cells upon activation and apoptosis. Agonists capable of inducing microparticle generation include cytokines, bacterial products, P-selectin, histamine. Cigarette smoke extract has also been recognized as an agonist involved in microparticle generation with an apoptosis-dependent mechanism. We investigated the possibility that cigarette smoke extract induces the rapid generation of proinflammatory microparticles by human mononuclear cells with a calcium-dependent mechanism.Materials and methodsHuman mononuclear cells were exposed to cigarette smoke extract. [Ca2+]i mobilization was assessed with the fluorescent probe Fluo-4 NW. Microparticles were quantified with a prothrombinase assay and by flow cytometry. Normal human bronchial epithelial cells and A549 alveolar cells were incubated with cigarette smoke extract-induced microparticles and the generation of ICAM-1, IL-8, and MCP-1 was assessed by ELISA.ResultsExposure to cigarette smoke extract induced a rapid increase in [Ca2+]i mobilization. Microparticle generation was also increased. EGTA, verapamil and the calmodulin inhibitor, W-7, inhibited microparticle generation. Incubation of lung epithelial cells with cigarette smoke extract-induced microparticles increased the expression of proinflammatory mediators.ConclusionsExposure of mononuclear cells to cigarette smoke extract causes a rapid shedding of microparticles with a proinflammatory potential that might add to the mechanisms of disease from tobacco use.

Aliskiren, a renin inhibitor, downregulates TNF-α-induced tissue factor expression in HUVECS

Angiotensin (Ang)II, the effector arm of the locally active renin—angiotensin system (RAS), modulates Tissue Factor (TF), the principal initiator of blood coagulation and a key promoter of atherothrombotic events. Consistent with that knowledge, previous data showed inhibitory properties of angiotensin-converting enzyme inhibitor (ACEI)s and angiotensin II type-1 receptor blocker (ARB)s, but no data are available about the effect of renin inhibition. We aimed to evaluate whether aliskiren, a direct renin inhibitor (DRI), modulates TNF-α-stimulated TF expression in cultured human umbilical vein endothelial cells (HUVECs). Zofenopril, an ACEI, and olmesartan, an ARB, were the controls. HUVECs were incubated with experimental drugs (1 nM) 30 min prior to TNF-α stimulation (0.1 ng/ml × 4 h). Main evaluation variables were procoagulant activity (single-stage clotting assay), TF antigen (ELISA) and mRNA expression (real-time polymerase chain reaction) in cell lysates. TNF-α stimulated procoagulant activity and increased TF antigen and mRNA expression. Aliskiren inhibited TNF-α-mediated TF stimulation; zofenopril and olmesartan exerted a comparable effect. We conclude that aliskiren, a DRI, downregulates TNF-α-stimulated TF expression in HUVECs, possibly as a reflection of endothelial renin activation by the cytokine.

ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 +/- 3 to 49 +/- 7% (SE). A significant increase from 17 +/- 4 to 39 +/- 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 ± 3 to 49 ± 7% (SE). A significant increase from 17 ± 4 to 39 ± 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin β-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

Angiotensin II induces the generation of procoagulant microparticles by human mononuclear cells via an angiotensin type 2 receptor-mediated pathway

INTRODUCTION Microparticles are small vesicles shed by cells upon activation and during apoptosis which participate in physiologically relevant phenomena, including blood coagulation. Intracellular calcium mobilization is one of the mechanisms of microparticle generation during cell activation. Because the renin-angiotensin system has been proposed as a link between hypertension and increased thrombotic risk, we investigated whether angiotensin II upregulates the generation of procoagulant microparticles by human mononuclear cells. MATERIALS AND METHODS Human mononuclear cells were exposed to angiotensin II for 15min. Intracellular calcium concentration was assessed by a Fluo 4 based kit. The supernatants were analyzed for both microparticle content, with a commercially available kit based on phosphatidylserine analysis, and microparticle-associated tissue factor, with a one-stage clotting assay. RESULTS Intracellular calcium concentration is increased upon exposure of mononuclear cells to angiotensin II. Incubation with angiotensin II stimulates microparticles release; microparticle-associated tissue factor is also upregulated. The effect is inhibited by an angiotensin receptor type 2 antagonist (PD123319) and not by two angiotensin type 1 antagonists (Losartan and Olmesartan). CONCLUSIONS Angiotensin receptor 2-mediated upregulation of tissue factor-bearing, procoagulant microparticle generation represents a novel mechanism linking the renin-angiotensin system to thrombosis.

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.