We have previously examined the ability of air pollution particles (PM10) to promote release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) from human peripheral blood mononuclear cells and demonstrated a role for calcium as a signaling molecule in this process. We have now studied the ability of oxidative stress induced by a synthetic oxidant tert-butyl hydroperoxide (tBHP) to induce TNF-α production via calcium signaling in the mouse macrophage cell line (J774). The oxidant tBHP significantly increased intracellular calcium and the release of TNF-α in J774 cells, an effect that was reduced to control levels by inhibition of calcium signaling with verapamil, BAPTA-AM, and W-7. This study also investigated interactions between PM10-treated macrophages and epithelial cells by using conditioned medium (CM) from PM10-treated mononuclear cells to stimulate the release of the neutrophil chemoattractant chemokine IL-8 from A549 lung epithelial cells. TNF-α protein release was demonstrated in human mononuclear cells after PM10 treatment, an effect that was inhibited by calcium antagonists. Treatment of A549 cells with monocyte/PM10 CM produced increased IL-8 release that was reduced with CM from monocyte/PM10/calcium antagonist treatments. The expression of ICAM-1 was increased after incubation with CM from monocyte/PM10 treatment, and this increase was prevented by treatment with CM from monocyte/PM10/calcium antagonist. These data demonstrate a link between oxidative stress, calcium, and inflammatory mediator production in macrophages and lung epithelial cells.
- lung macrophages
a considerable body of recent literature describes roles for reactive oxygen species (ROS) in the pathogenesis of pulmonary diseases such as acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma, and interstitial pulmonary fibrosis (11). An increased oxidative stress caused by enhanced release of ROS by air space leukocytes is also thought to occur in cystic fibrosis. ROS have the potential to cause direct injury to lung cells, and oxidative stress induced by ROS is also involved in enhancing inflammation through activation of redox-sensitive transcription factors such as NF-κB and AP-1 (15). The intracellular signaling molecule Ca2+ has been demonstrated to regulate the activation of transcription factors such as NF-κB by interacting with a number of proteins such as calmodulin and protein kinase enzymes (12) such as the calcium calmodulin-activated kinases (13). Although there is evidence that oxidative stress or ROS can activate calcium signaling and proinflammatory cytokine production, there is currently little evidence that ROS induce calcium signaling that is responsible for activating cytokine production. This study aimed to investigate this pathway and to determine its importance in controlling interactions between lung cells on exposure to pollution particles.
The production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is central to the regulation of inflammation and has been implicated in particle-related inflammation and pathology (18). Ultrafine or nanoparticles (particles with 1 dimension less than 100 nm) are one of the components of respirable particulate air pollution (PM10) that have been suggested to be responsible for the adverse respiratory and cardiovascular health effects associated with elevations of this pollutant (6). Much of the mass of PM10 is low in toxicity, and it has been suggested that combustion-derived nanoparticles are a key component that drives inflammation (8, 16). These nanoparticles are thought to be toxic due to their large surface area and surface reactivity (8).
Our (19) previous study using carbon nanoparticles has shown that these particles induce oxidative stress in lung cells as indicated by GSH depletion. Furthermore, we (1) have previously demonstrated that antioxidants were able to inhibit TNF-α production induced by the carbon nanoparticles, indicating a role for ROS in the induction of this cytokine expression. Other studies have also shown that nanoparticle carbon black and polystyrene beads stimulate an increase in intracellular Ca2+ and, in the case of carbon black, lead to the activation of transcription factors and TNF-α cytokine release (4, 7).
Various cell types including peripheral blood lymphocytes, macrophages, monocytes, and granulocytes are capable of producing proinflammatory cytokines such as TNF-α. Activation of the transcription factor NF-κB has been demonstrated in human peripheral blood mononuclear cells, airway epithelial cells, and lung tissue in response to oxidants or proinflammatory cytokines such as IL-1β and TNF-α (17). Interactions between particle-exposed macrophages and epithelial cells are thought to be modulated by such cytokines, but these interactions are poorly understood.
In this study, we have examined the effect of PM10 particles collected from the air and their ability to promote TNF-α release in human peripheral blood mononuclear cells. We also examined the role of calcium by including a range of calcium antagonists. The ability of oxidative stress to induce TNF-α release was studied in the mouse macrophage cell line (J774) using the synthetic oxidant tert-butyl hydroperoxide (tBHP) with and without calcium antagonists. We chose tBHP in this study because of its ability to activate various pathways (5, 14) and because it is more stable than other oxidants such as hydrogen peroxide. To investigate interactions between different cell types in the lung on exposure to PM10, the effects of conditioned medium (CM) obtained from particle-treated mononuclear cells were tested for their ability to stimulate release of the neutrophil chemoattractant IL-8 and to increase the expression of the adhesion molecule ICAM-1 in the human alveolar epithelial cell line, A549.
MATERIALS AND METHODS
J774 cell culture and treatment.
The murine macrophage cell line J774 was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 0.06 U/ml penicillin, and 30 μg/ml streptomycin (all obtained from Life Technologies) at 37°C in 5% CO2. Cells were scraped from the culture flasks and seeded at 0.5 × 106 cells/ml in 24-well plates and incubated for 24 h at 37°C before treatment. The medium was removed, and the cells were washed with RPMI and then treated with tBHP at a range of concentrations from 12.5 to 100 μM for 1, 2, and 4 h and with H2O2 at concentrations ranging from 10 to 500 μM for 2 h. In a separate series of experiments, tBHP at a final concentration of 12.5 μM was incubated in the absence and presence of a calcium antagonist, either verapamil (100 μM), W-7 (25 μM), or BAPTA-AM (25 μM). After incubation for 4 h at 37°C, the CM was removed, centrifuged at 1,200 g for 5 min, and stored at −80°C until required for TNF-α measurement by ELISA.
Measurement of murine TNF-α.
The TNF-α content of the J774 CM was quantified using a DuoSet sandwich ELISA system (R&D Systems) according to the manufacturer's instructions. Briefly, each well of a 96-well plate was coated overnight with capture antibody before washing with PBS containing 0.05% Tween 20 (PBS-Tween). Test CM was then added to the appropriate wells in triplicate. After incubation for 2 h at room temperature, the wells were washed, and a detection antibody was added for a further 2 h at room temperature. The wells were again washed with PBS-Tween, and horseradish peroxidase (HRP)-conjugated streptavidin was added to the wells before incubating for 20 min at room temperature. The color change was developed by adding peroxidase substrate to each well. The optical density of each sample was determined at a wavelength of 450 nm and a reference wavelength of 550 nm using an MRX plate reader (Dynatech).
Measurement of intracellular calcium.
J774 cells were scraped from culture flasks and adjusted to a density of 1 × 106 cells/ml in complete RPMI medium. Ten milliliters of this cell suspension was transferred to a universal tube and centrifuged at 900 g for 2 min, the medium was removed, and the cells were resuspended in 1 ml of PBS and transferred to an Eppendorf tube. The tube was centrifuged at 145 g for 2 min at 4°C, the PBS was removed, and the cells were resuspended in serum-free RPMI medium containing 23 mM HEPES buffer.
The J774 cells were then loaded with the fluorescent dye, fura-2 AM (2 μg/ml), to measure intracellular Ca2+. The tube was wrapped in foil and incubated in a shaking water bath for 20 min at 34°C to allow efficient loading of the fura-2 AM. After incubation, the tube was centrifuged at 145 g for 2 min, and the medium was removed and replaced with 1.5 ml of fresh, serum-free RPMI. The fura-2 AM-loaded cells were then transferred to a quartz cuvette, and the fluorescence was measured at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm with emission slits set at 5 nm using a LS-50B Luminescence spectrometer (Perkin Elmer, Buckinghamshire, United Kingdom). During the experiments, the cuvette temperature was kept constant at 37°C, and the cells were maintained in suspension using a magnetic stirrer.
Treatments (tBHP at 12.5 μM with and without the calcium antagonists at the concentrations previously described) were run for 2,000 s followed by stimulation with thapsigargin (10 nM) for 500 s. Thapsigargin was used to release the intracellular endoplasmic reticulum (ER) calcium stores as a method to assess cell viability. Depletion of the ER store, as indicated by a decreased response to thapsigargin, is indicative of events leading to apoptosis and cell death. Twenty microliters of 5% Triton X-100 solution was then added to the cuvette to lyse the cells and to allow the maximum fluorescence to be measured for a further 500 s. The minimum fluorescence was obtained by adding 15 μl of 0.5 M EGTA in 3 M Tris buffer to the cuvette for a final 500 s. The ratio of the fluorescence values at excitation wavelengths of 340 and 380 nm were calibrated and converted to Ca2+ concentration (nM) according to the protocol of Grynkiewicz et al. (10).
PM10 particle collection and characteristics.
The collection of PM10 samples was carried out by Casella Stanger (London, England). The site chosen for collection was Marylebone Road, London, England, an area that had high levels of traffic and consequently high levels of combustion-derived nanoparticles. Particles were collected onto Tapered Element Oscillating Microbalance (TEOM) filters.
Preparation of particles.
A PM10 filter was placed in a Bijou bottle, and 0.5 ml of PBS was added. After vortexing for 4 min to remove the particles, the suspension was transferred into a clean Bijou bottle. The concentration of particles in the suspension was assessed by densitometry. A series of ultrafine carbon black (UfCB; Printex 90, Degussa) particle dilutions ranging from 15.6 μg/ml to 1 mg/ml in PBS was prepared and sonicated for 5 min, and 75 μl of each concentration was added to triplicate groups of wells in a 96-well plate. The PM10 suspension (75 μl) was added into a separate triplicate group of wells. The concentration of PM10 particles in the sample was calculated from the linear regression of the UfCB standards after reading the absorbance at 340 nm on a Dynatec plate reader.
Isolation of human peripheral blood mononuclear cells.
Human peripheral blood mononuclear cells were prepared according to the protocol of Dransfield et al. (9). Ethical approval for the use of human donors was approved by Napier University's Ethics Committee. In brief, two separate volumes of 40 ml of blood were withdrawn from healthy consenting volunteers and transferred to 50 ml of sterile Falcon tubes containing 4 ml of 3.8% sodium citrate solution. Tubes were gently inverted and centrifuged at 250 g for 20 min, and the plasma was removed from each tube and pooled without disturbing the cell pellet. Dextran (Pharmacia), prepared as a 6% solution in saline, was warmed to 37°C before being adding to the cell pellet (2.5 ml/10 ml), and the volume was made up to 50 ml with sterile saline. Tubes were gently mixed, and the cells were allowed to sediment at room temperature for 30 min. To prepare autologous serum, calcium chloride solution (1 M, 220 μl/10 ml) was gently mixed with the plasma and incubated in a glass tube at 37°C until the clot retracted. Percoll (Pharmacia) gradients were made from a stock solution of 90% [18 ml of Percoll + 2 ml of 10× PBS (Life Technologies) without calcium or magnesium] to give final concentrations of 81, 70, and 55% using 1× PBS. The separating gradient was prepared by layering 2.5 ml of 70% Percoll over 2.5 ml of 81% Percoll. The leukocyte-rich fraction from the dextran sedimentation was transferred to sterile Falcon tubes, 0.9% saline was added to give a final volume of 50 ml, and the tubes were centrifuged at 250 g for 6 min. The pellet was resuspended in 55% Percoll, and 2.5 ml was layered over the previously prepared separating gradients. Tubes were centrifuged at 290 g for 20 min, and the mononuclear cells were collected from the 55/70 layer. Cells were washed twice with PBS, counted, and resuspended in RPMI medium at a concentration of 5 × 106 cells/ml, and 1 ml was added to each well of a 24-well plate. The cells were incubated for 1 h at 37°C, the medium was removed, replaced with RPMI plus 10% autologous serum, and incubated for 48 h at 37°C. After the second incubation, the medium was replaced, and the cells were incubated for a further 72 h before treatment.
Mononuclear cell treatment and CM.
Mononuclear cells were set up and differentiated as described above. Cells were treated with PM10 (10 μg/ml, 250 μl per well in RPMI medium without serum) for 4 h at 37°C containing calcium antagonists to give final concentrations of the calcium channel blocker verapamil (100 μM; Sigma), the calcium chelator BAPTA-AM (50 μM), the hydrophilic antioxidant Nacystelyn (200 μM; SMB Pharmaceuticals), and the lipophilic antioxidant Trolox (25 μM). After incubation, CM was removed, centrifuged at 12,000 g for 10 min, aliquoted, and stored at −80°C until required. A stock of PM10 and control mononuclear cell CM were also prepared for further experiments.
Treatment of mononuclear cell CM with TNF-α neutralizing antibody.
The PM10-treated mononuclear CM and controls prepared above were treated with TNF-α neutralizing antibody. Five microliters of antibody (1 mg/ml stock; Clone MAC 747, Serotec) were added to 250 μl of CM obtained from PM10-treated monocytes and incubated at 37°C for 2 h. In addition, the antibody was incubated with 250 μl of 400 pg/ml recombinant human TNF-α. The TNF-α content of the CM before and after antibody treatment was assessed using a TNF-α ELISA kit (Biosource).
Human TNF-α ELISA.
The CM previously prepared were assayed for TNF-α protein content using a commercially available human TNF-α kit (Biosource) according to the manufacturer's instructions. Briefly, each well of a 96-well plate was coated overnight with capture antibody before washing with PBS containing 0.05% Tween and then adding CM to the appropriate wells in triplicate groups. After incubation for 2 h at room temperature, the wells were washed, and a detection antibody was added and incubated for a further hour at room temperature. The wells were then washed with PBS-Tween before addition of HRP-conjugated streptavidin and incubated for 45 min at room temperature. Finally, the color was developed by adding peroxidase substrate to each well before reading the absorbance at 450 nm using a Dynatec plate reader.
A549 cell culture and treatments.
A549 epithelial cells were grown in continuous culture in RPMI 1640 medium (Sigma) containing l-glutamine and penicillin/streptomycin and 10% fetal calf serum. Cells were removed from culture by trypsinization and plated onto 24-well plates at 3 × 105 cells/ml (1 ml per well). Plates were incubated for 24 h at 37°C, wells were washed with PBS, and PM10/calcium antagonist-treated monocyte CM were added to each well in a 250-μl volume, including CM that had been pretreated with TNF-α neutralizing antibody. The plates were further incubated for 1 h at 37°C, and the CM were removed and retained for IL-8 analysis. In a separate series of experiments, PM10-treated mononuclear cell CM was added to each well, and at this point, the calcium antagonists were added directly to the A549 cells to give final concentrations described above. This experiment had two purposes: 1) to determine whether calcium signaling within epithelial cells was responsible for inducing IL-8 production, and 2) to determine whether the calcium antagonists present in PM10-treated monocyte CM were acting to prevent production of agents by monocytes that stimulate the epithelial cells or whether the inhibitors in the CM were acting directly on the epithelial cells preventing them from responding to the CM.
Human IL-8 ELISA.
Interleukin-8 was measured in CM according to the instructions of a commercially available kit (Biosource). Briefly, each well of a 96-well plate was coated overnight with capture antibody before washing with PBS containing 0.05% Tween and then adding test CM to the appropriate wells in triplicate groups. After incubation for 2 h at room temperature, the wells were washed, and a detection antibody was added and incubated for a further hour at room temperature. The wells were then washed with PBS-Tween, and HRP-conjugated streptavidin was added before incubating for 45 min at room temperature. Finally, the color was developed by adding peroxidase substrate to each well before reading the absorbance at 450 nm using a Dynatec plate reader.
Treatment and staining of A549 cells for flow cytometry.
A549 cells were set up as described above at a density of 5 × 105 cells per well in 12-well plates. The epithelial cells were incubated with 250 μl of CM from particle-treated monocytes for 18 h. In a separate series of treatments, CM from monocytes treated with PM10 were added directly to the A549 cells, and the calcium antagonists at the concentrations described previously were added at this point. Human TNF-α at concentrations ranging from 500 pg/ml to 10 ng/ml was also used to treat the A549 cells, and to investigate the role of TNF-α in the upregulation of ICAM-1, TNF-α neutralizing antibody was included at the same concentration used for previous experiments. Cells were removed from the wells by trypsinization as described above and washed twice with PBS before incubation for 1 h at room temperature with mouse anti-human ICAM-1 (Serotec) antibody diluted 1:100 in PBS. Cells were washed twice and incubated for 1 h at room temperature with FITC-labeled anti-mouse IgG (Sigma) diluted 1:200 in PBS. Cells were washed twice with PBS and analyzed using a Becton Dickinson flow cytometer. In addition to the treated cells, appropriate controls (+ve and -ve) for epithelial cell surface markers were included.
All data were examined using a general linear model with subsequent analysis of variance using Tukey's test using the Minitab statistical package.
Oxidant and calcium induced TNF-α release by J774 macrophages.
J774 cells were treated for 1–4 h with the oxidant tBHP at concentrations ranging from 12.5 to 100 μM. tBHP induced a negative dose response with the lowest concentration (12.5 μM), inducing the greatest significant increase in TNF-α release over and above the control. Higher doses were not effective due to toxicity (data not shown). From these experiments, a dose of 12.5 μM at a time point of 4 h was chosen. The experiment was repeated at this dose and time to include various calcium antagonists. These results are shown in Fig. 1, A and B. The oxidant significantly increased the release of TNF-α protein compared with the control levels (P < 0.01). When the Ca2+ channel blocker verapamil or the calcium chelator BAPTA-AM were included, the oxidant-induced TNF-α protein release was significantly inhibited. Cells treated with W-7 and tBHP did not generate more TNF-α protein release than the control (P > 0.05), but in this instance, the effect of the cotreatment was not significantly different from tBHP alone. Incubation of J774 cells with the oxidant H2O2 only produced an increase in TNF-α release at a concentration of 50 μM but was not significantly different from the control (Fig. 1C).
Effect of tBHP and verapamil on intracellular calcium levels.
Intracellular calcium concentration increased significantly when tBHP was added to the cells over 1,800 s (P < 0.05; Fig. 2). Values rose from 60.27 ± 6.61 nM (resting levels) to 230.38 ± 47.65 nM (12.5 μM tBHP). Treatment of the J774 cells with tBHP (12.5 μM) in the presence of verapamil significantly prevented the tBHP-induced elevation of Ca2+ in the cells from 230.38 ± 47.6 nM to 80.93 ± 6.5 nM (P < 0.05).
IL-8 release from A549 epithelial cells after treatment with monocyte CM.
Treatment of human primary monocytes with 10 μg/ml PM10 for 4 h induced release of TNF-α protein, and this effect was inhibited by the calcium antagonists verapamil and BAPTA-AM (Fig. 3), as demonstrated in our (1) previous study. However, the calmodulin inhibitor W-7 and the antioxidants did not suppress the PM10-induced TNF-α production. The CM from PM10-treated human monocytes stimulated significantly more IL-8 release from A549 epithelial cells compared with untreated cells (control, ∼300 pg/ml; PM10-treated, ∼950 pg/ml; Fig. 4). Addition of the calcium antagonists verapamil, BAPTA-AM, and W-7 or the antioxidant Trolox to the macrophages significantly reduced the ability of macrophage/PM10 CM to induce IL-8 release by the epithelial cells (P < 0.05). Nacystelyn addition to the macrophages did not significantly reduce the ability of macrophage/PM10 CM to reduce IL-8 release by A549 cells compared with macrophage/PM10 CM treatment alone.
Because of the possibility that the calcium antagonists and antioxidants added to the macrophages could affect A549 cell release of IL-8 rather than the production of factors by the macrophages that induce IL-8 release, the antagonists and the antioxidants were also added directly to the A549 cells. The addition of calcium antagonists added directly to A549 cells in combination with CM from mononuclear cells treated with PM10 particles alone is shown in Fig. 5. When PM10-treated monocyte CM were used to stimulate A549 cells in the presence of the calcium antagonists added directly to the A549 cells, there was no significant inhibition of CM-induced IL-8 release. Treatment of A549 cells with calcium antagonists or antioxidants alone did not stimulate IL-8 release (data not shown).
Incubation of monocyte CM with anti-TNF-α antibody before A549 epithelial cell treatment had no effect on IL-8 release (Fig. 6). These antibodies, however, were demonstrated to be effective at inhibiting recombinant TNF-α detection by the ELISA assay.
ICAM-1 expression in A549 epithelial cells.
Treatment of A549 epithelial cells with varying doses of TNF-α and subsequent detection of ICAM-1 expression is shown in Fig. 7A. Cells were treated for 18 h before treating with detection antibodies and analysis using flow cytometry. The results clearly indicate a dose-dependent effect of TNF-α on ICAM-1 upregulation. Analysis of untreated cells showed that ∼10% were ICAM-1 positive; at a dose of 1 ng/ml TNF-α, 50% of the cells were ICAM-1 positive. CM from monocytes treated with PM10 particles also increased the number of ICAM-1-positive cells (Fig. 7B). CM from control monocytes produced almost 40% ICAM-1-positive cells, which increased to 65% ICAM-1-positive cells on treatment with CM from PM10-treated monocytes (P < 0.05). Verapamil, BAPTA-AM, and W-7 treatment of the macrophages reduced the level of CM-induced ICAM-1 expression by the A549 cells so that it was no longer significantly different from the control. The calcium antagonists added to the A549 cells along with the macrophage CM did not prevent the increased expression of ICAM-1 (Fig. 7C). In the presence of the antioxidant Trolox, the PM10-treated macrophage CM no longer induced a significant increase in the percentage of cells expressing ICAM-1, whether the Trolox was added to the macrophages or epithelial cells. The antioxidant Nacystelyn had no effect in either experiment. In the presence of the TNF-α neutralizing antibody, the percentage of ICAM-1-positive cells was significantly reduced compared with TNF-α alone (Fig. 8).
Our (1, 3) previous studies demonstrate that both nanoparticles and PM10 induce calcium signaling in macrophages by a pathway involving ROS, leading to the production of TNF-α. The literature clearly shows that calcium signaling plays a role in controlling the production of proinflammatory mediators (7). However, there is little published evidence to suggest that oxidative stress can induce inflammation via calcium signaling.
In this study, we aimed to investigate whether the calcium-signaling pathways are involved in the release of TNF-α protein through oxidative stress by measuring [Ca2+] in J774 cells in response to various treatments. We have shown here that by treating J774 macrophages with the oxidant tBHP, [Ca2+] was increased in the macrophages to significant levels, suggesting a direct link between oxidative stress and Ca2+. This study demonstrated that [Ca2+] increased significantly following exposure to tBHP (12.5 μM), but when the J774 cells were cotreated with verapamil and tBHP (12.5 μM), the tBHP-induced elevation of [Ca2+] was prevented, suggesting that oxidative stress induces Ca2+ levels to increase in the cell via a mechanism involving opening of calcium channels. Verapamil inhibition alone is insufficient to conclude that calcium channel opening was enhanced, but our (18) previous studies using nanoparticle carbon black also demonstrate a similar response that was verified using calcium-free medium and Mn2+.
When J774 macrophages were treated with the oxidant tBHP at a concentration of 12.5 μM for 4 h, the oxidant stimulated a significant increase in the release of the proinflammatory cytokine TNF-α. The results of this study also suggest that the oxidant tBHP enhanced calcium signaling, leading to cytokine production. The Ca2+ antagonists verapamil and BAPTA-AM added to the cells in conjunction with tBHP also prevented an increase in tBHP-induced TNF-α protein release by the cells, suggesting that Ca2+ signaling in the cell cytoplasm has a direct effect on the release of proinflammatory cytokines by J774 macrophages thereby linking oxidative stress to the Ca2+-signaling pathway and inflammation.
A second oxidant H2O2 was also investigated. H2O2 (50 μM) induced a twofold increase in TNF-α release by the J774 cells after 2 h. However, this twofold increase was not statistically significant due to the instability of H2O2 and the presence of catalase in the cells. For these reasons, we chose to use tBHP for all subsequent experiments due to its more favorable stability.
This work supports our (2) previous work that demonstrated that ultrafine or nanoparticles stimulated nontoxic calcium-signaling events in macrophages via a mechanism involving ROS and that these changes led to increased TNF-α protein release. We (2) also demonstrated that the calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, and the calmodulin inhibitor W-7 all significantly inhibited the production of TNF-α protein release by nanoparticle carbon black-treated particles. In our (1) previous study, 14 nm of CB (100 μg/ml, 4 h) was found to increase TNF-α protein release to 110 pg/ml. In the present study, only 10 μg/ml of PM10 was required to induce a comparable response, suggesting a 10× difference in potency. In both studies, inhibition of calcium signaling with verapamil and BAPTA prevented the particle-induced TNF-α release. W-7, the calmodulin inhibitor, while significantly inhibiting TNF-α production induced by 14 nm of CB (1), did not significantly inhibit the PM10- or tBHP-induced cytokine expression, although it did prevent a significant increase. Furthermore, this might suggest that while calcium is clearly essential in driving the production of the proinflammatory cytokine in response to the oxidant and PM10, in contrast to the calcium-binding protein calcineurin, it may play a role, but it is not essential.
In our (19) study with lung epithelial cells, ultrafine but not fine carbon black particles were shown to interfere with metabolic processes via an oxidative stress mechanism, and changes in metabolic function may be linked to alterations in calcium homeostasis within the cell, although this has not been studied. Our (19) study using monomac-6 cells suggested that nanoparticles stimulated the entry of extracellular calcium, and treatment with thapsigargin further increased the cytosolic calcium ion concentration compared with fine particles. In the present study, peripheral blood mononuclear cells treated with PM10 particles were shown to release TNF-α, an effect that could be modulated with the calcium antagonists but not with the antioxidants. CM from these experiments were then used to treat A549 lung epithelial cells. The PM10-treated macrophage CM induced cytokine IL-8 release from the lung epithelial cell line. Additionally, the calcium antagonists and the antioxidant Trolox (but not Nacystelyn) added to the monocytes were able to modulate this effect, suggesting that calcium and ROS play a role in stimulating the release from macrophages of substances that activate epithelial cells. In contrast, the calcium antagonists and antioxidants when added to the A549 cells with the CM obtained from mononuclear cells treated solely with PM10 particles did not significantly inhibit IL-8 release. This confirms that the calcium antagonists and antioxidants added to the macrophages were inhibiting the PM10-induced production of mediators that activated the cytokine production by the epithelial cells rather than inhibiting the epithelial cells directly. Addition of particles directly to A549 epithelial cells presents problems. IL-8 released from these cells after stimulation can be adsorbed onto the particles themselves thereby making detection difficult (Stone et al., unpublished data).
Our data also suggest that TNF-α is not the main or sole stimulatory factor released from the mononuclear cells on stimulation with PM10 particles. Incubation of the PM10-treated monocyte CM with TNF-α neutralizing antibody had no effect on the ability of the CM to stimulate IL-8 release by A549 cells. This antibody was shown to be active in neutralizing recombinant human TNF-α and TNF-α present in the macrophage CM preventing its detection by ELISA. This suggests that factors other than TNF-α may be responsible for the increased IL-8 release by A549 cells, probably due to the redundant nature of the cytokine-signaling network. Our (3) previous study shows that PM10 induces IL-1 expression by monocyte-derived macrophages. IL-1 is a very potent inducer of inflammation and could be responsible for the CM activity.
Stimulation of epithelial cells with TNF-α or PM10-treated monocyte CM produced increased expression of the adhesion molecule ICAM-1 as assessed by flow cytometry. An interesting finding was that the increase in expression in epithelial cell ICAM-1 was reduced when the calcium antagonists were added to the macrophages along with PM10, suggesting a signaling role for calcium in the macrophages. Addition of calcium antagonists to the epithelial cells with CM from PM10-treated macrophages did not significantly reduce the amount of ICAM-1 expression. Increased adhesiveness of the epithelial surface of the lung plays a role in migration of macrophages. This, in turn, may be advantageous in clearing particle-laden cells from the lung. The antioxidant Trolox, but not Nacystelyn, prevented PM10-treated macrophage release of factors driving epithelial ICAM-1 expression, again suggesting a role for ROS in the proinflammatory effects of PM10. In general, it is worth noting that the lipophilic antioxidant Trolox was more effective than the hydrophilic antioxidant Nacystelyn at inhibiting the proinflammatory responses induced by the macrophages in this study. This may be because the lipophilic antioxidant protects the membrane of the cell against the impact of oxidants and particles. However, it is worth noting that in studies with carbon nanoparticles, Nacystelyn has prevented increased calcium signaling (18) and TNF-α protein production (2).
Taken together, the results of this study suggest that ROS stimulate Ca2+ signaling in macrophages leading to the production of proinflammatory cytokines. This effect is mimicked by PM10 (shown here) and nanoparticle treatment of macrophages. Calcium clearly plays a role in stimulating the PM10-treated macrophages to generate products that activate ICAM-1 and IL-8 expression in epithelial cells. The products made by macrophages that stimulate the epithelial cells remain unidentified, but this response does not appear to be driven by TNF-α alone. Further investigation into the link between oxidative stress, calcium, and the production of inflammatory mediators such as TNF-α may give a clearer understanding of the mechanism behind PM10- and nanoparticle-induced inflammation that could result in disease.
This project was generously funded by the Colt Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society