ToxSci Advance Access originally published online on August 11, 2008
Toxicological Sciences 2008 106(1):233-241; doi:10.1093/toxsci/kfn162
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Particle-Induced Cytokine Responses in Cardiac Cell Cultures—the Effect of Particles versus Soluble Mediators Released by Particle-Exposed Lung Cells
* Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, NO-0403 Oslo, Norway
Department of Pharmacology, University of Oslo, NO-0316 Oslo, Norway
1 To whom the correspondence should be addressed at Department of Air Pollution and Noise, Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway. Fax: +47-21076686. E-mail: Annike.Irene.Totlandsdal{at}fhi.no.
Received June 5, 2008; accepted August 5, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Increased levels of particulate matter have been associated with adverse effects in the respiratory as well as the cardiovascular system. The biological mechanisms behind these associations are still unresolved. Among potential mechanisms, particulate matter–associated cardiac effects may be initiated by inhaled small-sized particles, particle components and/or mediators related to inflammation that translocate into the pulmonary circulation. In the present study cytokine responses (interleukin [IL]-6, IL-1β, and tumor necrosis factor [TNF]-
) of primary rat cardiomyocytes and cardiofibroblasts in mono- and cocultures induced by direct exposure to particles, were compared with cytokine responses induced by mediators released by particle-exposed primary rat epithelial lung cells (conditioned media). Cells were exposed to a model ultrafine particle (ultrafine carbon black, Printex 90) and in selected experiments to an urban air particle sample (SRM 1648, St Louis, MO). In lung cell cultures both particle types induced release of IL-6 and IL-1β, whereas TNF- was only detected upon exposure to St Louis particles. The release of IL-6 by cardiac cells was strongly enhanced upon exposure to conditioned media, and markedly exceeded the response to direct particle exposure. IL-1, but not TNF-, seemed necessary, but not sufficient, for this enhanced IL-6 release. The role of IL-1 was demonstrated by use of an IL-1 receptor antagonist that partially reduced the effect of the conditioned media, and by a stimulating effect on the cardiac cell release of IL-6 by exogenous addition of IL-1 and IL-1β. These in vitro findings lend support to the hypothesis that particle-induced cardiac inflammation and disease may involve lung-derived mediators. Key Words: ultrafine particles; lung cells; cardiac cells; cytokines; cocultures.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In epidemiological studies increased levels of particulate matter (PM) in ambient air have been associated with adverse effects in the respiratory as well as the cardiovascular system (Pope and Dockery, 2006
). The explanation for the cardiovascular effects is not immediately apparent, and several pathways related to the mechanism of action have been suggested. Clinical studies have for instance indicated PM-induced disruptions of vascular function (Brook et al., 2002; Mills et al., 2005; Rundell et al., 2007). However, it is not clear whether particles induce sympathetic nervous system activation, or create an inflammatory response with oxidative stress, leading to an imbalance between vasoconstrictive and vasodilative mediators (Rundell et al., 2007). Although the vascular system presumably is an obvious target for PM-induced cardiovascular responses, the cardiac tissue should also be taken into account when assessing potential underlying mechanisms. As revealed by in vivo studies with rats and mice, PM-induced effects in the myocardium may include decreased numbers of granulated mast cells, multifocal myocardial degeneration, chronic-active inflammation, and fibrosis (Kodavanti et al., 2003), as well as myocardial oxidative stress and increased myocardial infarction size after ischemia-reperfusion injury (Cozzi et al., 2006). Inflammation is considered a key event in the development of the adverse health effects associated with PM exposure, but exactly how and where in the cardiopulmonary system inhaled particles elicit such effects, is still unclear. Inhaled particles may exert effects indirectly in cardiac tissue via systemic release of proinflammatory mediators from the lung, and/or directly if particle components and/or small-sized particles cross the air-blood barrier. Once inflammatory mediators, particles or particle components enter the circulation, contact with cardiac tissue may occur directly in the cardiac chambers and through the coronary circulation.
A number of studies have indicated the potential of inhaled ultrafine (< 100 nm) particles (Chen et al., 2006; Kreyling et al., 2002; Nemmar et al., 2002; Oberdorster et al., 2002; Takenaka et al., 2001, 2004) or particle components (Wallenborn et al., 2007) to cross the air-blood barrier and to reach secondary target organs including the heart, but the extent of translocation is still under discussion (Mills et al., 2006). In addition to the direct interaction between potentially translocated particles and cardiac tissue, particle-induced pulmonary inflammation and accompanying release of proinflammatory mediators into the blood circulation, represents a plausible mechanism for PM-induced myocardial injury. Lung-derived mediators may affect blood constituents or blood vessels, promote an autonomic stress response possibly leading to altered heart rhythm, or induce inflammatory responses in extrapulmonary organs (Donaldson et al., 2005). In support of this, cytokines known to be released by particle-exposed human lung cells in vitro, were also detected in the blood of subjects during an episode of acute atmospheric air pollution (van Eeden et al., 2001). However, the challenge remains to reveal to what extent cytokines detected systemically are released from the pulmonary system.
We have recently demonstrated that particles have the potential to induce release of interleukin [IL]-6 and IL-1β in cultures of primary rat cardiac cells (Totlandsdal et al., 2008). These cytokines are among the cytokines known to be elevated in heart failure (Anker and von Haehling, 2004; Colucci, 1997; Lisman et al., 2002; Torre-Amione, 2005) and these have also been detected in the blood of subjects exposed to increased levels of particulate air pollution (Tornqvist et al., 2007; van Eeden et al., 2001). Myocardial cells are generally categorized into muscle cells termed cardiomyocytes (CMs), which may account for approximately 30–40% of total cell numbers (Vliegen et al., 1991), and nonmyocytes mainly consisting of endothelial cells and cardiofibroblasts (CFs) (Camelliti et al., 2005). The cardiac cell cultures used in our previous (Totlandsdal et al., 2008) and in the present study, consisted of mono- and contact cocultures of CMs and CFs.
In this study it was assumed that lung-derived inflammatory mediators, in addition to particles, are able to cross the air-blood barrier and to reach the heart. We hypothesized that mediators released by particle-exposed lung cells, had the potential to induce cytokine responses in cardiac cell cultures. Our experimental approach was to expose primary rat epithelial lung cells to particles and subsequently expose cardiac cell cultures to supernatants collected from these cells, after removal of the particles (conditioned media). For comparison, experiments of cardiac cell cultures exposed directly to particles were also included. Because the cytokines IL-1β and tumor necrosis factor [TNF]- may activate the production of IL-6 in cardiac cells (Loppnow et al., 2001; Turner et al., 2007), we have also attempted to determine the role of these cytokines in the release of IL-6 from cardiac cells exposed to conditioned media.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemicals/reagents.
Williams' E medium without glutamine (for culture of epithelial lung cells) was purchased from Bio Whittaker (Walkersville, MD) and Joklik Modified Minimum Essential Medium (MEM) (M0518) (for culture of cardiac cells), NaHCO3, MgSO4, D,L-carnitine, creatine, taurine, penicillin-streptomycin, CaCl2, trypsin (T-4665), bovine serum albumin (BSA), insulin, hydrocortisone, transferrin, epidermal growth factor (EGF), sodium selenite, glutathione, protease, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), recombinant IL-1
and IL-1β from Sigma-Aldrich (St Louis, MO). Fatty acid free BSA (A6003) was used for heart perfusion and in the cardiac cell culture medium, and normal fraction V BSA (A9647) in the exposure medium. Antibiotics used in culture medium of lung cells included penicillin-streptomycin purchased from Lonza (Verviers, Belgium) and ampicillin and fungizone obtained from Bristol-Myers Squibb AB (Bromma, Sweden). Collagenase type 2 was purchased from Worthington Biochemical Corporation (Freehold, NJ), laminin from Invitrogen (Carlsbad, CA), fetal bovine serum (FBS) from EuroClone (Pero, Italy), gentamicin from Aventis Pharma A/S (Lysaker, Norway), and IL-1 receptor antagonist Kineret (also Anakinra) from Amgen (Thousand Oaks, CA).
Particles.
Pigment black Printex 90 obtained from Degussa Corporation (Frankfurt, Germany) was used as a model for ultrafine combustion particles. The primary particle size of Printex 90 has been estimated as 12–17 nm (Stoeger et al., 2006), but in culture medium most of these primary particles appear as parts of aggregates of different sizes. Experiments were also carried out with commercially available St Louis particles (SRM 1648), a standard reference material of urban PM, collected in St. Louis, MO (National Institute of Standards & Technology, Gaithersburg, MD). Particles were suspended in cell exposure medium (2 mg/ml) and stirred overnight before exposure of cells.
Isolation of primary rat epithelial lung cells.
Epithelial lung cells were isolated from lungs of pentobarbital-anesthetized (10 mg/kg, i.p.) adult male WKY/NCrl rats weighing 220–300 g, as previously described (Låg et al., 1996). In short, the lungs were perfused via the pulmonary artery with a calcium- and magnesium-supplemented phosphate buffer. Macrophages were removed by lavage before the lungs were treated with protease (1.4 mg/ml), minced and filtered. Subsequently, populations of predominantly epithelial cells were isolated by centrifugal elutriation and differential attachment. These cells were suspended in Williams' medium E supplemented with insulin (5 µg/ml), hydrocortisone (0.087 µg/ml), transferrin (5 µg/ml), EGF (10 ng/ml), sodium selenite (6.2 ng/ml), glutathione (5 µg/ml), ampicillin (100 µg/ml), streptomycin (100 µg/ml), fungizone (0.25 µg/ml), HEPES (15mM), and 5% heat-inactivated FBS.
Culture of primary rat epithelial lung cells.
Isolated lung cells were plated in 35 mm six-well culture dishes (surface area per well approximately 10 cm2) at a density of 4,000,000 cells per well in Williams’ E medium with supplements. Before use cell cultures were incubated for approximately 2 days at 37°C in a humidified atmosphere of 5% CO2 in air. Five hours prior to particle exposure, medium was replaced by exposure medium as used for cardiac cells (see below).
Isolation of rat ventricular CMs and CFs.
Hearts from isofluran-anesthetized adult male Crl:WI (Han) rats (250–300 g) were excised, placed into ice-cold saline and mounted in a Langendorff perfusion apparatus. Subsequently, ventricular CMs were isolated by enzymatic digestion using trypsin (60 U/ml) and collagenase (90 U/ml), as described previously with some modifications (Viko et al., 1995). Briefly, the hearts were aorta-perfused at 6.4 ml/min with a Joklik's MEM solution supplemented with 24 mmol/l NaHCO3, 1.6 mmol/l MgSO4, 1 mmol/l D,L-carnitine, 10 mmol/l creatine, and 20 mmol/l taurine. After 2 min of perfusion, BSA (0.1%) and trypsin (60 U/ml) were added to the perfusion medium. Collagenase (90 U/ml) was introduced 6 min later. After 26 min of perfusion CaCl2 was added (0.25 mmol/l). After 12 additional min the hearts were removed from the perfusion device and cut into small pieces, after removal of the atria. The tissue was dispersed in medium as used in the final step of the perfusion, and placed in a shaking water bath for 10 min (110 strokes/min, 37°C) and subsequently filtered through a nylon mesh (pores 250 µm), before the CMs were separated from the CFs by repeated centrifugations (36 x g, 3 and 4 min). The resulting pellet was dispersed and allowed to sediment twice (2 x 20 min) in medium as used in the first step of perfusion, but supplemented with BSA (1%) and CaCl2 (0.5 mmol/l), for removal of trypsin and collagenase. The fibroblast-containing supernatant was further centrifuged at higher speed (1500 x g, 5 min).
Culture of CMs.
Freshly isolated CMs were suspended in cardiac cell culture medium (Joklik's-MEM solution supplemented with 24 mmol/l NaHCO3, 1.6 mmol/l MgSO4, 1 mmol/l D,L-carnitine, 10 mmol/l creatine, 20 mmol/l taurine, 0.2 mmol/l CaCl2, 0.1% BSA, and 1% penicillin-streptomycin) and plated in 35 mm six-well culture dishes (Falcon, BD Biosciences, Le Pont de Claix, France), 120,000 cells/well, precoated with laminin (10 µg/ml). After a 2-h incubation period (37°C, 5% CO2) the medium was replaced by exposure medium (cardiac cell culture medium with 1% BSA) for removal of nonattached cells and acclimatization overnight prior to particle exposure.
Culture of CFs.
Freshly isolated CFs were suspended in cell culture medium without BSA, but supplemented with gentamicin (40 µg/ml) and heat-inactivated FBS (10%), and plated on 100 mm culture dishes (Corning Inc, Corning, NY) and incubated at 37°C with 5% CO2. After 2 h, medium was refreshed in order to remove non-attached cells. Before use CFs were incubated for another 7–9 days with medium refreshment every 2–3 days, and passaging before reaching confluence. One day prior to particle exposure CFs were plated into 35 mm six-well culture dishes (240,000 cells per well). After 2 h of incubation allowing cell attachment, culture medium was replaced by exposure medium as used for the CMs for removal of nonattached cells and acclimatization overnight prior to exposure. Culture dishes with CF monocultures were, in contrast to CM-containing cell cultures, not precoated with laminin. For CF monocultures 2-h incubation with medium supplemented with 10% FBS was considered sufficient to facilitate cell attachment (Totlandsdal et al., 2008).
Preparation of cocultures of CMs and CFs.
In case of contact cocultures, freshly isolated CMs (120,000 per well) were plated on laminin-coated wells preplated with CFs (240,000 per well). Further treatment was identical to CM monocultures (see above).
Exposure of cell cultures.
After medium refreshment, lung and cardiac cell cultures were exposed to various concentrations of Printex 90 (0, 50, 100, 200, or 400 µg/ml) or St Louis particles (0, 200 µg/ml) for 20 h. Conditioned media were collected and centrifuged twice for removal of dead cells (300 x g) and particles (8000 x g), and stored at –70°C until cytokine analysis or exposure of cardiac cells to conditioned media from lung cells. To control for the possible effects of incomplete removal of particles from conditioned media, experiments also included exposure of cardiac cells to medium subjected to cell-free incubation with particles for 20 h and subsequent centrifugation. In transfer experiments cardiac cells were exposed to 500 µl fresh medium and 1 ml conditioned media. Where applicable, cardiac cell cultures were treated with 500 µl of culture medium containing IL-1 receptor antagonist (45 µg/ml) for 30 min before 1 ml conditioned media was added (resulting in a final antagonist concentration of 15 µg/ml). An overview of the experimental set-up is presented in Figure 1.
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Quantification of cytokine release.
Release of IL-6, IL-1
, IL-1β and TNF- into the culture medium was measured using enzyme-linked immunoabsorbent assay (ELISA) (R&D Systems, Minneapolis MN), according to the manufacturer's manual. The increase in color intensity was measured and quantified using a plate reader (TECAN Sunrise, Phoenix Research Products, Hayward, CA) with software (Magellan V 1.10). Cytokine concentrations in culture wells (1.5 ml) are expressed in ng/ml.
Calculations and statistical analysis.
Results are expressed as means ± SEM of separate replicate experiments, where each experiment was carried out with cells from separate isolations from rat lungs or hearts. For each result presented, the specific number of experiments, which always is at least three, is stated in the figure legends.
Where appropriate, concentration-response curves (Fig. 2) were constructed according to Ariëns et al. (1964), by estimating centiles (i.e., EC10 to EC100 for each dose-response experiment) and calculating the corresponding means. This was done by a computer program based on linear interpolation between responses observed experimentally. This calculation provides mean curves expressing the response as a percentage of the maximal response with biologically proper horizontal positioning and slope. These responses were recalculated into, and expressed as, absolute levels of cytokine release. Horizontal positioning of the curves was expressed as EC50 indicating the concentration resulting in half-maximal effect.
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4) of values observed in separate experiments, points with horizontal bars represent means ± SEM of EC50 values, all other points represent average percentiles of the maximal response obtained by linear interpolation between responses observed experimentally.The data in Figures 3, 4, and 5 was analyzed statistically by application of a two-way ANOVA with Bonferroni post tests. Statistical analysis of the data presented in Figure 3 (absence vs. presence of IL-1 receptor antagonist [IL-1ra]), Figures 4 and 5 was carried out on log-transformed data. Statistical analyses were performed using GraphPad Prism software (version 4.03, Inc., San Diego, CA). p < 0.05 was considered to reflect statistically significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Printex 90–Induced Release of IL-6 and IL-1β in Epithelial Lung Cell Cultures
Exposure of lung cells to Printex 90 particles (0–400 µg/ml, 20 h) induced concentration-dependent releases of both IL-6 and IL-1β (Figs. 2A and 2B). IL-6 levels increased from 0.15 ± 0.07 ng/ml to 2.9 ± 1.0 ng/ml, with an estimated EC50 between 40–53 µg/ml of Printex 90. IL-1β levels increased from 0.015 ± 0.005 to 0.15 ± 0.05 ng/ml, and the estimated EC50 was between 65 and 148 µg/ml. Printex 90 did not seem to induce the release of TNF-
at any concentration tested (data not shown).
Cytokine Responses of Cardiac Cells to Printex 90 versus Conditioned Media from Printex 90–Exposed Epithelial Lung Cells
Conditioned media from particle-exposed lung cells (0–400 µg/ml, 20 h) induced an enhanced release of IL-6 in all cardiac cell cultures compared with direct particle exposure (Figs. 2C–E). The greatest contrast in response was noted in the CF monocultures. In those cultures a direct Printex 90-induced release of IL-6 could not be detected and the detected IL-6 levels in control cultures of approximately 2 ng/ml were not exceeded. In contrast, conditioned media from unexposed and exposed lung cells caused an elevation in IL-6 release in CF monocultures from 6.2 ± 2.6 to 24 ± 12 ng/ml (Fig. 2C). In CM monocultures direct exposure to Printex 90 increased IL-6 levels from 1.3 ± 0.5 to 4.7 ± 1.5 ng/ml, in contrast to the increase from 6.6 ± 1.6 to 22 ± 6 ng/ml, when exposed to conditioned media from unexposed and exposed lung cells (Fig. 2D). IL-6 levels in cocultures increased from 43 ± 12 to 80 ± 34 ng/ml and from 53 ± 8 to 139 ± 35 ng/ml, following direct exposure to particles or to conditioned media from unexposed and exposed lung cells (Fig. 2E), respectively. Notably, basal levels were increased in all cardiac cell cultures exposed to conditioned media, but relatively less increased in cocultures compared with monocultures. Exposure to control medium, only subjected to incubation with and subsequent removal of particles, did not alter IL-6 levels. This indicates that the responses elicited by conditioned media were not caused by the unintended presence of particles. Estimated EC50s in CM- and cocultures exposed to conditioned media (between 25–36 and 35–50 µg/ml, respectively) were apparently lower than in CM- and cocultures exposed directly to Printex 90 (between 40–96 and 51–89 µg/ml, respectively).
Transfer of conditioned media from lung cells to cardiac cells did not cause any further increase in the IL-1β levels detected after exposure of lung cells to particles, or induce a release of TNF- in the cardiac cell cultures (data not shown).
Effect of IL-1ra on IL-6 Release in Cardiac Cell Cultures Exposed to Conditioned Media from Printex 90–Exposed Epithelial Lung Cells
Because IL-1β may activate the production of IL-6 (Loppnow et al., 2001), and because this cytokine was detected after exposure of the lung cells to particles, IL-1β may be a factor contributing to the enhanced release of IL-6 detected in cardiac cell cultures exposed to conditioned media from the lung cells. To elucidate this, the effect of treating the cardiac cells with IL-1ra prior to and during exposure to the conditioned media, was tested (Fig. 3). However, treatment with IL-1ra may, in addition to IL-1β, also reduce the effects of the closely related cytokine IL-1, as IL-1 and IL-1β binds to the same membrane receptor (Boraschi and Tagliabue, 2006). Analysis of supernatants from lung cells used in this set of experiments revealed comparable Printex 90-induced release of IL-1 and IL-1β (data not shown). Treatment with IL-1ra reduced the levels of IL-6 in both mono- and cocultures of cardiac cells exposed to conditioned media from particle-exposed lung cells (Figs. 3A–C). The inhibitory effect of IL-1ra treatment on IL-6 release seemed most evident in the cocultures. In cocultures, the elevation in IL-6 levels caused by exposure to conditioned media from lung cells exposed to increasing concentrations of Printex 90, was apparently eliminated (Fig. 3C). In CF and CM monocultures IL-1ra treatment proved less effective and significant particle concentration-dependent responses remained (Figs. 3A and 3B). IL-1ra significantly lowered the basal release of IL-6 in CF mono- and in the coculture, which was induced by exposure to conditioned media from unexposed lung cells (Figs. 3A and 3C).
Comparison of Cardiac Cell IL-6 Release Induced by Exposure to Conditioned Media and Exposure to IL-1 Cytokines
The reduced IL-6 release from cardiac cells with IL-1ra prior to and during exposure to conditioned media, may indicate a role for IL-1 cytokines in the IL-6 response. To further elucidate the role of IL-1 and IL-1β in this response, the capability of exogenous addition of these cytokines to induce release of IL-6 in cardiac cell cultures was compared with the responses of cardiac cells to conditioned media from the lung cells. At IL-1 and IL-1β concentrations well above IL-1β levels detected in the conditioned media (Fig. 2B), consistently lower IL-6 levels were detected than after exposure to conditioned media (Figs. 4 A–C). This difference in IL-6 levels appeared smaller in cocultures than in monocultures (Figs. 4A–C), indicating that IL-1 and IL-1β may play a greater role in the response of cocultures to conditioned media. Furthermore the experiments revealed equal capabilities of IL-1 and IL-1β to induce release of IL-6 in cardiac cell cultures. A similar pattern was also observed when significantly higher concentrations of IL-1 and IL-1β were added (data not shown).
The Role of TNF- in the IL-6 Responses of Cardiac Cells to Conditioned Media
St Louis particles were included in the present study due to their ability to induce release of TNF-, in addition to IL-6 and IL-1β, in lung cell cultures (Figs. 5A–C). Compared with conditioned medium from unexposed lung cells, conditioned medium from lung cells exposed to St Louis particles only very slightly enhanced the release of IL-6 from cardiac cell cultures (Figs. 5D–F). Despite the contrast in release of TNF- between lung cells exposed to St Louis particles and Printex 90, conditioned medium from lung cells exposed to St Louis particles did not induce greater release of IL-6 than conditioned medium from Printex 90-exposed lung cells in any of the cardiac cell cultures (Figs. 5D–F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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We have recently reported that ultrafine Printex 90 particles have the potential to induce expression and release of IL-6 in cardiac cells in vitro, using mono- and cocultures of CMs and CFs (Totlandsdal et al., 2008
). In the present study we observe that the Printex 90 particles induce the release of mediators from lung cells with the potential to strongly enhance the release of IL-6 from cardiac cell cultures. Notably, this response was of much larger magnitude than the IL-6 response observed in cardiac cells upon direct exposure to equivalent particle concentrations. Furthermore, our data suggest that IL-1, probably in concert with other factors, may play an important role in this enhanced response. These in vitro findings lend support to the hypothesis that particle-induced cardiac inflammation and disease may involve lung-derived mediators.
Extrapolated to the in vivo situation, our in vitro findings may suggest that particle-induced lung-derived mediators are more important for cardiac inflammatory responses than inhaled particles that may reach the cardiac tissue via the systemic circulation. The short distance between the lung and heart favors that both lung-derived mediators and particles that have entered the pulmonary circulation very rapidly reach the heart, before being distributed to other tissues. However, ultrafine particles have a tendency to aggregate at high concentrations. This may enhance the uptake of inhaled ultrafine particles by alveolar macrophages (Takenaka et al., 2001) and potentially reduce translocation of particles into the pulmonary circulation. In contrast, particle-induced synthesis and release of inflammatory mediators by lung cells may be less affected by particle aggregation. Surface area estimates of Printex 90 particles indicate that a relatively large particle surface area remains despite aggregation (Renwick et al., 2004). The particle surface area is considered an important parameter in the toxicity and the proinflammatory potential of ultrafine particles (Brown et al., 2000; Oberdorster, 2001). This may implicate that acute cardiac responses to lung-derived mediators are likely to occur at much lower inhalation concentrations of particles than cardiac responses to translocated particles. However, it should be emphasized that both plausible pathways may be involved in the in vivo situation, and that they also may interact. Recently, an in vivo study, quantifying the extrapulmonary translocation of intratracheally instilled particles in rats, indicated a major role for pulmonary inflammation in enhancing the translocation of particles (Chen et al., 2006).
An intriguing issue is to identify the soluble factor(s) released from Printex 90–exposed and unexposed lung cells responsible for the enhanced release of IL-6 from the cardiac cells. Although several mediators may be involved, the present study suggests lung-derived IL-1 and IL-1β to be important mediators. We observed that, in addition to IL-6, both IL-1 (data not shown) and β were induced by exposure of the lung cells to Printex 90, and that treatment with IL-1ra reduced the release of IL-6 from cardiac cell cultures exposed to conditioned media from the Printex 90–exposed lung cells. The inhibitory effects of IL-1ra on the release of IL-6 is in line with findings from studies with rat hearts, indicating that inhibition of IL-1 may attenuate inflammatory responses in the myocardium (Suzuki et al., 2001). The role of IL-1 in the release of IL-6 by cardiac cells was further investigated by exposing cardiac cell cultures to IL-1 and β. Both IL-1 and β proved able of inducing release of IL-6 in cardiac cultures, but the levels were well below IL-6 levels detected upon exposure to conditioned media. The concentrations of IL-1 and IL-1β (200 and 400 pg/ml) used in these experiments were well above the levels detected in the conditioned media and exposure to even significantly higher concentrations of these cytokines did not notably reduce the difference to conditioned media. This indicates that there are mediators, in addition to IL-1, contributing to the enhanced release of IL-6 by cardiac cells exposed to conditioned media. These mediators may also contribute by enhancing the effect of IL-1. It may be speculated whether these mediators act via the IL-1 receptor, or via other receptors activating parallel intracellular signaling pathways involved in the transcription and release of IL-6 and/or upregulation of IL-1 receptors.
Another cytokine potentially involved is TNF-. Similar to IL-1, TNF- is known to induce the release of other cytokines, including IL-6 (Turner et al., 2007). As reported in the present study, lung cells exposed to St Louis particles, but not to Printex 90, responded with an increase in TNF-, whereas both particles types induced release of IL-6 and IL-1β. However, our data revealed comparable IL-6 responses of cardiac cells to conditioned media from lung cells exposed to the different particles, suggesting that the additional TNF- induced by St Louis particles, did not augment the IL-6 response. This indicates that TNF- plays a minor role, if any, compared with IL-1 in the response of cardiac cells to conditioned media from particle-exposed lung cells. However, the possibility that St Louis particles contain a factor inhibiting an effect of TNF- cannot be excluded. Among possible mediators involved in the enhanced release of IL-6 from cardiac cells exposed to conditioned media from lung cells, IL-6 itself may also be a candidate, considering that IL-6 also may stimulate its own synthesis. In further investigation of heart cell responses to lung-derived mediators, the role of IL-6 should also be evaluated. Furthermore, it would be interesting to carry out a screening of the conditioned media in order to identify additional mediators that are likely to be involved.
The effect of IL-1ra proved most efficient in cocultures of cardiac cells, in which the elevation in IL-6 levels caused by exposure to conditioned media from lung cells exposed to increasing concentrations of Printex 90, seemed to be completely eliminated. Furthermore, the relative effect of exogenous addition of IL-1 and IL-1β, compared with the effect of conditioned media from particle-exposed lung cells, was apparently larger in cocultures than in the monocultures. The reason for this discrepancy in responses between mono- and cocultures is not clear. One possible explanation may be that contact between CM and CF in cocultures causes increased expression of IL-1 receptors compared with monocultures. Upregulation of several genes involved in inflammatory responses, including IL-1 receptors, has been detected in a coculture model consisting of other cell types (Popovici et al., 2006), but to what extent this can be extrapolated to our coculture model remains to be determined. Another possible explanation may be that several of the additional mediators involved in the enhanced IL-6 response of cardiac cells to conditioned media are already present in the cocultures.
Because our experimental model system does not closely resemble an in vivo situation, our data should be carefully interpreted. An important question is to what extent only lung-derived cytokines contribute to levels in the pulmonary veins. Notably, also the cells of the vessel wall and the heart are known to produce IL-1 and respond to this mediator by production of other cytokines or regulation of other cardiovascular functions (Loppnow et al., 2001). The diversity of different cells present in the cardiopulmonary system capable of producing overlapping mediators makes it challenging to determine to what extent the lung is an important source. In the applied model all possible mediators released by the lung cells were transferred directly to the cardiac cell cultures. In vivo, the production, release and transport of mediators are likely to be significantly influenced by interactions with other cells and tissues before reaching the cardiac tissue.
This study provides information about the potential importance of lung-derived mediators in stimulating IL-6 responses in isolated cardiac cells. Further studies are needed to examine whether similar mechanisms operate in the heart and predispose for development of cardiac disease. Whether and to what extent lung-derived mediators, including IL-1, are released from the lung into the circulation at particle concentrations that may be achieved in the lungs during realistic in vivo exposure scenarios, remains to be determined. It has previously been demonstrated that cytokines, including IL-6 and IL-1β may alter mechanical activity in CMs (Pathan et al., 2004; Roberts et al., 1992). Furthermore, a study with neonatal CMs indicates that exposure to metals associated with particles may alter spontaneous beat rate as well as the expression of ion channels and sarcolemmal proteins relevant to electrical remodeling and slowing of spontaneous beat rate (Graff et al., 2004). This indicates that it may be of relevance to also include mechanical responses of CMs in further characterization of cardiac cell responses to particles or mediators from particle-exposed lung cells.
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FUNDING |
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Research Council of Norway.
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ACKNOWLEDGMENTS |
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At the Norwegian Institute of Public Health, we would like to thank Tonje Skuland, Edel Lilleaas, and Hans Jørgen Dahlman for isolation of primary lung cells and Prof. Erik Dybing for feedback on the manuscript. At the Department of Pharmacology, University of Oslo, Iwona Schiander is thanked for isolation of primary cardiac cells.
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