Chapter 3: Rapid shedding of MP by human mononuclear cells exposed to Cigarette Smoke Extract (CSE)

Chapter 3:

Rapid shedding of MP by human mononuclear cells exposed to

Cigarette Smoke Extract (CSE)

3.1 CSE and vascular diseases

Tobacco smoke is a worldwide epidemic currently considered the most important preventable cause of death in the Western Countries [52]. Cigarette smoke (CS) consists of about 4700 different compounds, known to be antigenic, cytotoxic, mutagenic and carcinogenic [53]. These substances are dispersed in the gas phase. The particles phase contains important constituents of CS such as tar, nicotine, aromatic hydrocarbons, phenol and cresol. The mean size of these particles is 0.1-0.5 μm, so they are able to reach the small airways. CS can induce a strong pro-oxidative effect in smokers by different ways, either by direct measurements of the oxidative burden (ROS production by peripheral blood cells) or by the effects of oxidative stress on target molecules (lipid peroxidation products and oxidized proteins) or as the responses to the oxidative stress (decreased concentrations of antioxidant scavengers) [54]. Although the mechanisms whereby cigarette smoke causes disease are complex and not fully understood yet, CS affects multiple organ systems resulting in numerous co-called tobacco-related diseases. Chronic Obstructive Pulmonary disease (COPD) is a syndrome characterized by progressive airflow limitation caused by chronic inflammation of the airways and lung parenchyma. CS can cause pulmonary oxidative stress, a condition that characterizes patients with COPD and that is thought to contribute to chronic pulmonary inflammation. It has been shown that CS stimulates the local inflammation. Several studies demonstrated that the exposure to CS of cultured human mononuclear cells increases the production of pro-inflammatory chemokines and cytokines, such as IL-8, IL-6, granulocytes-macrophage colony-stimulating factor (GM-CSF) and TNF-α [55]. The release of pro-inflammatory and pro-chemoattractant molecules is not limited to immune cells, but also structural lung cells such as pulmonary fibroblasts, and both bronchial and alveolar epithelial cells, release chemoattractants for neutrophils, in particular IL-8,

upon CS exposure [55]. Furthermore, CS seems to decrease the protection and repair mechanisms of the epithelial barrier. CS is one of the main risk factor leading to atherosclerosis. The systemic inflammation induced by CS on macrophages and lymphocytes and the consequent release of proinflammatory cytokines, cause endothelial cell activation and vascular damage which promote the prothrombotic state and atherosclerosis plaque production [56]. TF is widely express in atheromatous plaque and its contact with blood containing FVIIa is thought to produce a strong thrombogenic stimulus that it’s critical to the development of coronary thrombosis after plaque rupture. It has been showed that carotid andarterectomy plaques from 28 smokers and 28 age- and sex-matched nonsmokers, the former were reported to have increased TF expression and activity [57]. Indeed, the circulating form of TF have an important role in homeostasis and thrombosis. High levels of TF have been demonstrated in patients at risk of thrombotic disorders. A recent study comparing 10 smokers and 15 nonsmokers, the former had significantly higher plasma TF activity and levels increased further 2 hours after smoking [58]. Recent clinical evidences have suggested MP as important mediators involved in vascular damage. Heiss C e coworkers demonstrated that after a brief exposure to secondhand smoke (SHS) the EMP plasma concentration increases significantly and remains elevated 24h after the exposure. Moreover, in vitro data showed that SHS can stimulate the endothelial cells to generate MP [59]. A recent study demonstrated that an increase in EMP with apoptotic characteristics in smokers with normal spyrometry but reduced DLCO, suggesting that the pulmonary

emphysema is linked to the capillary endothelium apoptosis and that it can be monitored at the early stages with the measurement of EMP [60]. Li and coworkers have recently demonstrated that CSE induces the generation of apoptotic procoagulant MP from human mononuclear cells [61] in a process requiring the activation of the apoptotic process.

3.2 Aim

The aim of this work was to investigate whether CSE can induce the liberation of procoagulant MP by mononuclear cells and to inquire if the rapid intracellular calcium mobilization was involved in this process. Moreover, we focused on the proinflammatory potential of MP generated through this mechanism on the epithelial cells of the airways.

3.3 Matherial and Methods

3.3.1 Reagents and kits

RPMI 1640 medium, high glucose-DMEM, penicillin, streptomycin, L-glutamine, fetal bovine serum (FBS), trypsin/EDTA, trypsin inhibitor, trypan blue, PBS, HEPES, sodium chloride, Ficoll-Hypaque, dextran, sodium citrate, calcium chloride, calcium ionophore A23187 (A23187), ethylene glycol tetraacetic acid (EGTA), Ham’s F12, DNAse, formaldehyde, o-phenylendiamine, peroxidase-conjugate anti-mouse IgG (γ-chain specific) and propidium iodide were obtained from Sigma (Milan, Italy). Vitrogen was obtained from Tebubio (Milan, Italy). BEGM Bullet Kit was obtained from Cambrex (Caravaggio, BG, Italy). Thromboplastin standard was obtained from Beckman Coulter (Milano, Italy). Human anti-TF antibody was obtained from America Diagnostica (Instrumentation Laboratory, Milano, Italy). The Zymuphen MP-Activity kit was obtained from Hyphen BioMed (Neuville-sur-Oise, France). The Fluo-4 NW Calcium Assay kit was obtained from Molecular Probes (Invitrogen, Milan, Italy). Annexin V-FITC was obtained fromAlexis (Vinci Biochemicals, Firenze, Italy). Human IL-8 matched antibody pairs were obtained from Bender Medsystems GmbH (Vienna, Austria). Human MCP-1 Elisa Ready-Set-Go! was obtained from eBioscience (Ireland, United Kingdom). The mouse anti-human-ICAM-1 monoclonal antibody (clone 15.2) and the mouse anti-human-IgG1 isotype control were obtained from

Ancell (Bayport, MN, USA). All other chemicals were obtained from the hospital pharmacy and were of the best grade available.

3.3.2 Cell culture

Cells of the human alveolar epithelial line, A549 (American Type Culture Collection, CCL-195), were kindly provided by Dr. R. Danesi, University of Pisa, Italy. A549 cells were maintained in RPMI supplemented with 10% (vol/vol) FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin in a humidified 95% air- 5% CO2

atmosphere at 37°C. Normal human bronchial epithelial cells (NHBEC) were obtained from subjects undergoing diagnostic bronchoscopy as previously described [62]. Briefly, afterthe patient's informed consent to the procedure was received,the fiber-optic bronchoscope was positioned at the level of thecarina and/or the level of second- or third-order bronchial branchings.The use of local anesthetics was kept as low as possible to minimize their effects on cell viability. Four to six brushings of grosslynormal bronchial mucosa were obtained. The cells werethen removed from the brush by vortexing in Ham's F-12 medium-10%FBS. The cells were brought to the laboratory in ice and incubated with DNase (50 µg/mL) to eliminate clumping. After a wash withice-cold, serum-free Ham's F-12 medium, the cells were resuspendedin BEGM and plated on Vitrogen 100-coated culture flasks. Indirect immunofluorescence with anti-cytokeratin antibodiesconfirmed the epithelial origin of the cells [62]. Cells used in the specific experiments reported here were obtained from a patient with peripheral lung cancer undergoing diagnostic bronchoscopy and were harvested from the contralateral main bronchus. The cells were used at passages 3-4.

3.3.3 Mononuclear cells isolation and culture

Mononuclear cells were isolated either from fresh buffy coats obtained from the local blood bank or from the peripheral blood of normal volunteers as described [63]. Briefly, a fresh buffy coat was diluted 1:1 with sodium citrate 0.38% in normal saline solution, mixed gently with an equal volume of 2% Dextran T500, and left for 30 minutes for erythrocyte sedimentation. The leukocyte-rich supernatant was recovered and centrifuged for 10 minutes at 200 x g. The pellet was resuspended in 30 mL of sodium citrate solution, layered over 15 mL of Ficoll-Hypaque and centrifuged for 30 minutes at 350 x g at 4°C. The mononuclear cell-rich ring was recovered and washed twice in sodium citrate solution. Mononuclear cells were then

resuspended in RPMI/10% FBS and allowed to adhere for 18 hours at 37°C on 24-well plates (2x106 cells/well) or on 10-cm Petri dishes (40x106 cells/dish).

3.3.4 CSE

Camel cigarettes (R.J. Reynolds Tobacco Company) which contain 10 mg tar and 0.8 mg nicotine/cigarette, were used. CSE was prepared by a modification of the method of Carp and Janoff as previously described [64]. Briefly, a 5 cm cigarette without filter was combusted with a modified syringe-driven apparatus, the smoke was bubbled through 25 ml of serum-free RPMI and the resulting solution was considered 100% CSE. The resulting suspension was filtered through a 0.22-μm pore filter to remove bacteria and large particles. To ensure the reproducibility among different CSE batches, the absorbance measured at 340 nm was used as a measure of the “strength ”of the extract. Dilutions were made with RPMI to obtain the desired absorbance. Different concentrations of CSE (from 2.5% to 10%) were applied to mononuclear cells cultures within 30 min of preparation.

3.3.5 MP generation and purification

Mononuclear cells were washed three times with pre-warmed serum-free RPMI. For MP generation, CSE or A23187 (12 µM) were added; after 15 min at 37°C the supernatants were recovered, cleared by centrifugation at 14,000 x g for 5 min at room temperature to remove dead cells and big cell fragments that might have detached during the stimulation, and immediately used for further experiments. In selected experiments, MP (12 mL) were further purified by ultracentrifugation (100,000 x g for 2 hours, 4°C); the pellet was resuspended in 250 μL of normal saline and used in a one-stage clotting assay to measure TF-dependent coagulation.

3.3.6 Measurement of MP

PS-positive MP in each sample were detected using the Zymuphen MP-activity kit (Hyphen BioMed, Neuville-sur-Oise, France) according to the manufacturer’s instructions and expressed as PS equivalents (nMPS).

3.3.7 Measurement of intracellular calcium concentration

Molecular Probes Fluo-4 NW Calcium Assay kit was used to measure the changes in the intracellular calcium concentration ([Ca2+]i) of mononuclear cells. Pre-washed

mononuclear cells on 96-multiwell plate (0.5 x 106 cells/well) were loaded with 100 μL of the dye loading solution containing Fluo-4 NW dye and probenecid, according to the manufacturer’s instructions. The 96-well plate was incubated at 37 °C for 45-60 min in the dark and fresh CSE (10%) and A23187 (12 µM) as a positive control were added to the cells. The changes in Fluo-4 NW fluorescence were measured by the Wallac 1420 Victor 2 (PerkinElmer, Milan, Italy) at λex 494 nm and λem 516 nm.

Calcium mobilization was observed over time (up to 80 sec) and analyzed by the Wallac 1420 Software version 3 (PerkinElmer Life and Analytical Sciences, Wallac, Milan, Italy). The increase in [Ca2+]i fluorescence (expressed as relative fluorescence

units, RFU) was calculated as the difference between the mean stimulation fluorescence value and the mean baseline fluorescence observed (Δ RFU).

3.3.8 Detection of apoptosis

Apoptosis was measured using annexin V-FITC as marker of apoptosis and propidium iodide (PI) as marker of necrotic cells according to a standard protocol. In brief, mononuclear cells (4x106 cells/mL) were grown on Petri dishes and treated with CSE 10% for 15 min. After treatment, 5x105 cells were resuspended in 100 µL of annexin-V binding buffer containing 5 µL fluorescein isothiocyanate (FITC) conjugated annexin-V (50 µg/mL). After a short incubation in the dark at room temperature, 5 µL of propidium iodide (50 µg/mL) was added followed by the binding buffer. The samples were then analyzed in a FACScalibur flow cytometer (Beckton Dickinson, San Jose, CA, USA) to differentiate apoptotic cells (early apoptosis: annexin-V positive

and PI negative, lower right quadrant; late apoptosis: annexin-V positive and PI positive, upper right quadrant) from necrotic cells (annexin-V negative/PI positive, upper left quadrant). Ten thousand events were recorded for each treatment group.

3.3.9 ELISA for IL-8 and MCP-1 detection

MP generated by mononuclear cells were sedimented by ultracentrifugation (100,000 x g for 2 hours, 4°C) and quantified by the Zymuphen MP-Activity kit. Resuspended MP were adjusted to 3.5 nM PS and incubated with A549 and NHBEC grown to confluence in 96-well plates for 24 h at 37°C. Following an 18-hour incubation, the conditioned medium was harvested, cleared by centrifugation for 5 minutes at 12,000 x g, and analyzed for IL-8 and MCP-1 content. IL-8 and MCP-1 in supernatants from A549 and NHEBC epithelial cells was measured by a sandwich ELISA kit according to the manufacturer’s instructions.

3.3.10 Measurement of cell surface ICAM-1 expression

ICAM-1 expression on HEBC and A549 cells was measured by direct cell ELISA as described with minor modifications [62]. Briefly, cells were seeded at 20x103 cells/well in 0.1 ml of medium in 96-well plates. When the cells reached subconfluence, they were washed three times with PBS and fixed with 3.7% formaldehyde (w/v). Following additional extensive washing, nonspecific Ab binding sites were blocked by incubating the cells with PBS/3% BSA (w/v) for 2 h. Cells were then incubated with mouse anti human ICAM-1 Ab (5 mg/ml) in PBS 1% BSA (w/v) for 2 h at 37°C, then washed three times with wash buffer. Peroxidase-conjugated antimouse IgG Ab was then added at the concentration recommended by the manufacturer and incubated for 1 h at 37°C. After extensive washing, bound enzyme was determined by adding o-phenylendiamine, 1 mg/ml in 50 mM sodium citrate (pH 4), containing 0.015% H2O2. After blocking the reaction with 2 M H2SO4, the plate

was read in a spectrophotometer (Titertek Multiskan MCC ELISA reader; Flow Laboratories, McLean, VA) at 492 nm.

3.3.11.Assessment of MP-bound TF activity

TF activity was measured in MP generated in vitro from human mononuclear cells by a one-stage clotting time assay as described [65], except that the normal human plasma was made MP-poor by ultracentrifugation (100,000 x g for 2 hours, 4°C). The results were expressed in arbitrary units (AU) of procoagulant activity by comparison with a standard curve obtained using a human brain thromboplastin standard. This preparation was assigned a value of 1000 UA for clotting time of 30 sec. An anti-human TF Antibody (30 µg/mL) was used to assess the specificity of the test.

3.3.12 Data presentation and statistical analysis

Unless otherwise indicated, data are shown as mean ± SEM from n independent, consecutive experiments. Comparisons among groups were made by ANOVA for repeated measures followed by Bonferroni’s analysis using Graph Pad Prism Software version 4.00 for Windows (GraphPad Software, San Diego, Caliphornia, USA). Values of p < 0.05 were considered statistically significant).

3.4 Results

3.4.1 CSE induces the mobilization of [Ca2+]i in mononuclear cells

Mononuclear cells were loaded with Fluo-4-NW probe and stimulated with fresh CSE. Figure 3.1a shows that CSE induces a rapid increase in [Ca2+]i. A23187 (12 µM) was

Fig. 3.1 [Ca2+]i mobilization in human mononuclear cells induced by CSE (a) and A23187 (b) as assessed by Fluo4-NW incorporation. Data from one experiment representative of 3.

3.4.2 CSE induces the generation of MP by mononuclear cells

To investigate whether CSE treatment induces a rapid release of MP following calcium mobilization, mononuclear cells were stimulated with fresh CSE at different dilution (2.5%, 5%, 10%) for 15 min. Figure 3.2b shows that mononuclear cells generate MP upon exposure to CSE for 15 minutes in a concentration-dependent manner; the effect reaches statistical significance at 10% CSE (fig.3.2a).

Fig. 3.2 CSE induces the generation of MP by human mononuclear cells. a) Human mononuclear cells were incubated with CSE (10%) for 15 minutes and MP generation was assessed. N = 8; * p<.05 by paired t-test. b) Concentration-response curve. Data from one experiment representative of 6.

3.4.3. Rapid CSE-induced generation of MP by mononuclear cells is dependent on calcium mobilization

As shown in figure 3.3, preincubation of cells with the calcium chelator EGTA (1 mM for 30 min) largely inhibited CSE-induced MP generation, confirming a role for calcium mobilization in the phenomenon.

Fig. 3.3 Rapid CSE-induced generation of MP by mononuclear cells is dependent on calcium. Cells were exposed to CSE in the absence or in the presence of EGTA (1 mM) and MP generation was assessed. N = 3; * p<.05 by Anova analysis with Bonferroni post test .

3.4.4 Rapid CSE-induced generation of MP in not apoptosis dependent

To exclude the involvement of apoptosis in the generation of MP in our experimental settings, mononuclear cells were analyzed by FACS following annexin V/PI staining. The results obtained confirmed that CSE treatment did not induce apoptosis in the short time frame of our experimental conditions (fig. 3.4).

Fig. 3.4 Analysis of apoptosis in human mononuclear cells upon incubation with CSE (10%). Data from one experiment representative of 2.

3.4.5. CSE-induced MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells.

Numerous studies have demonstrated the proinflammatory potential of LMP [1, 4, 10, 15, 20]. In the present study we investigated whether CSE-induced MP are able to upregulate the synthesis of proinflammatory mediators by lung epithelial cells.

A549 and NHBEC cells were incubated with purified CSE-induced MP for 18 h and IL-8 and MCP-1 was measured in the supernatants. Expression of membrane bound ICAM-1 was assessed by c-ELISA. Figures 3.5-7 show that CSE-induced MP upregulate the expression of IL-8 and ICAM-1 by both cell lines, and of MCP-1 by A549 cells.

Fig. 3.5 IL-8 expression by lung epithelial cells exposed to CSE-induced MP. A549 (a) or NHBEC (b) were incubated for 24 h with MP (3.5 nM PS) shed by human mononuclear cells upon incubation with 10% CSE for 15 minutes. IL-8 was then measured in the conditioned medium. N=5 for A549 and 9 for NHBEC; * p<0.05 by paired t-test.

Fig. 3.6 MCP-1 expression by A549 cells exposed to CSE-induced MP. A549 cells were incubated for 24 h with MP (3.5 nM PS) shed by human mononuclear cells upon incubation with 10% CSE for 15 minutes. MCP-1 was then measured in the conditioned medium. N=7; *p<0.05 by paired t-test.

Fig. 3.7 ICAM-1 expression by lung epithelial cells exposed to CSE-induced MP. A549 (a) or NHBEC (b) were incubated for 24 h with MP (3.5 nM MP) shed by human mononuclear cells upon incubation with 10% CSE for 15 minutes. Membrane bound ICAM-1 was then measured by c-ELISA. N=4 for A549 and 3 for NHBEC; * p<0.05 by paired t-test.

3.4.6 CSE-induced MP express TF on their surface

To evaluate whether MP induced upon short exposure of mononuclear cells to CSE express TF on their surface, we analyzed TF procoagulant activity of purified MP released by treated and untreated cells through a one-stage clotting test. As shown in Figure 3.8, CSE treatment induces a 2.6-fold, significant increase in MP bound TF activity. A monoclonal antibody to TF (30 µg/mL) inhibited most of the procoagulant activity (not shown), confirming the identity of this activity with TF.

Fig. 3.8 MP-bound TF shed by human mononuclear cells upon incubation with CSE. Human mononuclear cells were incubated with CSE (10%) for 15 minutes. Shed MP were purified by ultracentrifugation and analyzed for TF activity with a one-stage clotting assay. N=8; * p<0.05 by paired t-test.

3.5 Discussion

Originally considered laboratory artifacts or cell debris devoid of physiological significance, MP have recently become the object of intense studies, since their role

in human physiology and pathophysiology has been clearly recognized. In the field of respiratory diseases, Bastarache and coll. have recently investigated the potential role of MP in the pathogenesis of the acute respiratory distress syndrome, demonstrating a significant increase in MP concentration, likely of epithelial origin, in the edema fluid of patients with the syndrome compared with patients with hydrostatic edema [66]. An increase in circulating MP of endothelial origin was observed in cigarette smokers with early lung destruction (normal spirometry and reduced diffusion capacity), compared to “healthy” smokers and normal controls [60]. Several pathophysiologically relevant agonists have been shown to induce the shedding of MP, including cytokines, such as TNF-α [9], bacterial products, such as LPS [67], histamine [63], P-selectin [68]. Of note, the composition and activity of MP differ not only based on the cell from which they originate, but also on the stimulus used for their induction [67]. We postulated that MP induced by CS , one of the most important causes of lung disease, are proinflammatory. The present data confirm that MP generated by human mononuclear cells in a calcium dependent fashion upon exposure to CSE have a proinflammatory potential. The role of IL-8, MCP-1, ICAM-1 and, more broadly, of inflammation in lung diseases is well recognized [69], [70], [71]. Thus, CSE induced MP might contribute to mount the inflammatory response that characterizes these diseases.

Because they were detected through their annexin V binding properties, MP are procoagulant by definition, since binding to annexin V takes place via PS, an essential component of the prothrombinase and tenase complexes. However, we also investigated the presence of TF on the MP surface. TF is the initiator of the so-called extrinsic pathway of blood coagulation. The current data extend Li’s observation of the presence of TF on MP generated via an apoptosis-dependent mechanism and confirm that MP originated by mononuclear cells upon exposure to CSE express fully functional TF and potentially contribute to the pathogenesis of cardiovascular diseases. In conclusion, our data demonstrate that exposure of mononuclear cells to CS causes rapid cell activation, via intracellular calcium mobilization, and shedding of MP with proinflammatory and procoagulant potential. Taken together with Li’s data, the results indicate that CSE has the potential to stimulate MP generation by two

distinct mechanisms, one rapid, [Ca2+]idependent, and one more slow, the requires

the induction of apoptosis. Modulation of MP generation might represent in the future a viable approach to treat smoke-induced lung and vascular diseases.