Calcium and ROS-mediated activation of transcription factors and TNF-{alpha} cytokine gene expression in macrophages exposed to ultrafine particles

D. M. Brown,1 K. Donaldson,1 P. J. Borm,2 R. P. Schins,2 M. Dehnhardt,2 P. Gilmour,3 L. A. Jimenez,4 and V. Stone1

1School of Life Sciences, Napier University, and 4ELEGI Laboratory, University of Edinburgh, Edinburgh, United Kingdom; 2Particle Research Core, Institut für Umweltmedizinische Forschung, Düsseldorf, Germany; and 3Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, North Carolina

Submitted 5 May 2003 ; accepted in final form 1 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultrafine (Uf) particles are a component of particulate air pollution suggested to be responsible for the health effects associated with elevations of this pollutant. We have previously suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals. Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease. The effect of Uf particles on rat alveolar macrophages and human blood monocytes was investigated with reference to the roles of calcium and reactive oxygen species (ROS). TNF-{alpha} protein release, intracellular calcium concentration, TNF-{alpha} mRNA expression, and transcription factor activation were studied as end points after treatment of rat alveolar macrophages or peripheral blood monocytes. The calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, the calmodulin inhibitor W-7, and the antioxidants Trolox and Nacystelin (NAL) were included in combination with Uf particles. Verapamil reduced intracellular calcium concentration in rat alveolar macrophages on stimulation with Uf particles. This effect was also apparent with transcription factor AP-1 activation. All antagonists and antioxidants reduced Uf-stimulated nuclear localization of the p50 and p65 subunits of NF-{kappa}B in human monocytes. Verapamil, BAPTA-AM, and NAL reduced Uf-stimulated TNF-{alpha} protein release, whereas only verapamil reduced Uf-stimulated mRNA expression in rat alveolar macrophages. In human monocytes, verapamil, Trolox, BAPTA-AM, and W-7 reduced Uf-stimulated TNF-{alpha} protein release. These findings suggest that Uf particles may exert proinflammatory effects by modulating intracellular calcium concentrations, activation of transcription factors, and cytokine production through a ROS-mediated mechanism.

reactive oxygen species; tumor necrosis factor-{alpha}


ULTRAFINE (UF) PARTICLES (particles with a diameter <100 nm) are one of the components of respirable particulate air pollution (PM10) suggested to be responsible for the health effects associated with elevations of this pollutant (10). In addition, exposure to Uf particles in the workplace could be a significant problem because of their wide use in the manufacture of a diverse array of products (38). We (10, 47) and others (53) have suggested that Uf particles, through the induction of oxidative stress, may induce inflammation in the lung, thus exacerbating preexisting illness in susceptible individuals.

In high-dose inhalation studies, Uf particles of various types have been shown to cause lung inflammation, increased chemokine expression, epithelial cell hyperplasia, pulmonary fibrosis, and even lung tumors (7, 14, 18, 33, 34). Alveolar macrophages are considered to play a key role in particlemediated inflammation and lung disease.

The genes that encode many cytokines, such as TNF-{alpha}, are under the control of the redox-sensitive inflammationrelated transcription factors NF-{kappa}B and activator protein 1 (AP-1), and expression of proinflammatory mediators in a number of diseases, such as sepsis, is driven by intracellular calcium-related signaling pathways (15, 41, 44). Activation of the proinflammatory transcription factor NF-{kappa}B is regulated via a number of second messengers, including calcium (8) and reactive oxygen species (ROS) (40). On stimulation of the cell by specific agonists, calcium is released from the endoplasmic reticulum stores, which leads to a calcium ion influx across the plasma membrane via calcium channels (23). Several pathogenic particles and oxidants (17, 27, 49) have been reported to cause changes in calcium ion flux within the cell, so there may be a role for calcium homeostasis in the proinflammatory effects of Uf particles. In addition, intracellular calcium has been implicated in the control of a large variety of cellular processes, including superoxide anion generation via NADPH oxidase (39), nitric oxide production by constitutive nitric oxide synthetase activity (39), cytoskeletal function (56), secretion of proteins (3), and activation of transcription factors such as NF-{kappa}B and nuclear factor of activated T cells (8). Hence, there is a large number of physiological and pathological cellular functions that could be stimulated via calcium signaling after exposure to Uf particles. We have previously demonstrated that Uf particles stimulate calcium influx into macrophages and that they enhance agonist (thapsigargin)-induced calcium signaling events that could be inhibited by antioxidants, indicating the involvement of oxidative stress (49, 51).

This study aimed to investigate the effect of Uf particles on TNF-{alpha} expression by primary macrophages and to elucidate the role of intracellular calcium signaling events and oxidative stress in the control of this cytokine.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Particle characteristics. Fine carbon black (CB) was obtained from H. Haeffner (Huber 900; Chepstow, UK), and Uf carbon black (UfCB) was obtained from Degussa (Printex 90). The average particle size of the two types of particle was 260 nm for CB and 14 nm for UfCB. The surface area of UfCB compared with CB is orders of magnitude higher, which may be an important consideration in these experiments. The characteristics and details of these particles have been published previously (50).

Bronchoalveolar lavage. Care and animal procedures were approved by Napier University Ethics Committee. Male Wistar rats, ~3 mo old, were killed with a single intraperitoneal injection of pentobarbitone. The lungs were then cannulated, removed, and lavaged with 4 x 8-ml volumes of sterile saline. The bronchoalveolar lavage (BAL) was centrifuged at 900 g for 2 min at 4°C, the supernatant was removed, and the resultant cell pellet was resuspended in 1 ml of MEM supplemented with 0.1% BSA and penicillin/streptomycin (Sigma, Poole, Dorset, UK). The total cell number was counted, and cytocentrifuge smears were prepared, which were stained with Diffquick (Raymond A. Lamb, London, UK) for differential cell counts.

Isolation of human peripheral blood mononuclear cells. Human peripheral blood mononuclear cells were prepared according to the protocol of Dransfield et al. (13). In brief, two separate volumes of 40 ml of blood were withdrawn from healthy volunteers and transferred to 50-ml sterile Falcon tubes containing 4 ml of 3.8% sodium citrate solution. Tubes were gently inverted and centrifuged at 1,350 rpm for 20 min. 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 added to the cell pellet (2.5 ml/10-ml cell pellet), 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 (220 µl 1 M/10 ml) was gently mixed with the plasma and incubated in a glass tube for 1 h at 37°C. Percoll (Pharmacia) gradients were made from a stock solution of 90% (18 ml of Percoll + 2 ml of 10x PBS; Life Technologies) (without calcium or magnesium) to give final concentrations of 81, 70, and 55% using 1x PBS. The separating gradient was prepared by layering 2.5 ml of 70% Percoll over 2.5 ml of 80% 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 1,250 rpm for 6 min. The pellet was resuspended in 55% Percoll, and 2.5 ml were layered over the previously prepared separating gradients. Tubes were centrifuged at 1,798 rpm 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 1640 medium at a concentration of 8 x 106 cells/ml, and 1 ml was added to each well of a 12-well plate. The cells were incubated for 1 h at 37°C, and the medium was removed and replaced with RPMI 1640 plus 10% autologous serum and incubated for 48 h at 37°C. After the second incubation, the medium was replaced, and the cells incubated for a further 72 h before treatment.

Cell treatment. Primary rat alveolar macrophages were resuspended in MEM (0.1% BSA) at a concentration of 1 x 106 cells/ml, and 1 ml was added to each well of a 24-well plate. Cells were incubated for 1 h at 37°C before being washed twice with 1-ml volumes of PBS. Primary human monocytes were isolated and plated as described above and allowed to differentiate for 5 days before use. After being washed, the rat or human cells were incubated with 1 ml of medium containing treatments for 4 h at 37°C. The various treatments consisted of either MEM alone or medium containing either UfCB or CB at a final concentration of 200 µg/ml. The calcium channel blocker verapamil (100 µM; Sigma), the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)AM (50 µM), the hydrophilic antioxidant Nacystelin (200 µM; SMB Pharmaceuticals), and the lipophilic antioxidant Trolox (25 µM) were also included with and without UfCB particles.

In a separate series of experiments, cells were treated with UfCB at concentrations ranging from 12.5 to 100 µg/ml. After being incubated for 4 h at 37°C, the medium was removed and centrifuged at 12,000 g for 5 min to remove residual particles. The supernatants were finally transferred to sterile Eppendorf tubes, which were stored at -80°C until required for biochemical analysis.

mRNA extraction. The experiments described were also used to generate cells for total RNA extraction. After the supernatant was removed, 600 µl of TRI reagent (Sigma) were added to each well. The lysed cells were then scraped from the surface of the plate using a cell scraper and transferred to Eppendorf tubes. Two hundred microliters of chloroform were added to each Eppendorf, and the cells were vortexed for 15 s and were allowed to stand at room temperature for 15 min. The resulting mixture was centrifuged at 12,000 g for 15 min at 4°C. The colorless upper phase was transferred to a fresh Eppendorf before 450 µl of isopropanol were added. The mixed samples were allowed to stand for a further 10 min at room temperature. Again, the tubes were centrifuged at 12,000 g for 10 min at 4°C, the supernatant was removed, and the RNA pellet was washed in 1 ml of 75% ethanol. The resulting samples were then vortexed briefly, centrifuged at 7,500 g for 5 min at 4°C, and the RNA pellet was air-dried for 10 min. The RNA was then suspended in 50 µl of diethylpyrocarbonate-treated water and stored at -70°C until required for quantification and RT-PCR.

RT-PCR. The RT-PCR procedure was carried out using the Promega Access kit. Briefly, a master mix of the kit reagents was prepared according to the manufacturer's instructions. Ten microliters of RNA at 0.03 µg/ml was added to 40 µl of the master mix, containing 10 µl of the appropriate rat primers GAPDH 5'-ATG ACT CTA CCC ACG GCA AG-3', 5'-CCA CAG TCT TCT GAG TGG CA-3', or TNF-{alpha} 5'-TAC TGA ACT TCG GGG TGA TTG GTC C-3', 5'-CCA AGA GAA GTT CCC TCT TCC GAC-3' (MWG AG Biotech, Ebersberg, Germany). Tubes were placed in a thermal cycler programmed for the following temperatures and times. Program 1 was 48°C for 45 min; program 2 was 94°C for 2 min; and program 3 was 95°C for 30 s, 60°C for 1 min, and 68°C for 2 min. Programs 2 and 3 were repeated for 25 cycles for GAPDH and 30 cycles for TNF-{alpha}. Program 4 was run at 68°C for 7 min, followed by cooling to 4°C. The resulting RT-PCR products were separated by electrophoresis using a 2% agarose gel containing 1 µg/ml of ethidium bromide and viewed under ultraviolet light. The RT-PCR bands were quantified by densitometry using Syngene software, and the TNF-{alpha} band intensity was expressed as a percentage of the corresponding GAPDH band. These results were then expressed as a percentage of the untreated control.

TNF-{alpha} ELISA. The supernatants previously prepared were assayed for TNF-{alpha} protein content using a commercially available rat TNF-{alpha} ELISA kit (ImmunoKontact, Liverpool, UK) or human TNF-{alpha} kit (Biosource) according to the manufacturer's instructions. Briefly, each well of a 96-well plate was coated overnight with capture antibody before being washed with PBS containing 0.05% Tween; then test supernatant was added to the appropriate wells in triplicate groups. After being incubated for 1 h at room temperature, the wells were washed, and a detection antibody was added and incubated for 1 further h at room temperature. The wells were then washed with PBS/Tween, and horseradish peroxidase-conjugated streptavidin was added before being incubated for 1 h 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.

Calcium imaging. Rat alveolar macrophages were isolated from BAL, as previously described, by adhesion onto 26-mm glass coverslips contained in six-well plates. Cells were seeded in RPMI 1640 medium containing 0.1% BSA and penicillin/streptomycin at a density of 5 x 105 cells/ml and incubated at 37°C, 5% CO2 for 1 h before being washed with 1 ml of PBS. Before particle treatment and digital enhanced video microscopy (Roper Scientific), cells were loaded with the calcium-sensitive dye fura 2-AM (2 µg/ml in RPMI 1640; Sigma) for 30 min at 37°C. The coverslips were washed with PBS and assembled into the microscope holder, and 400 µl of RPMI 1640 medium without phenol red (Sigma) were added. The fluorescence ratio was observed (excitation 340 and 380 nm, emission 510 nm) at a magnification of x63 (Zeiss Axiovert microscope). Images were captured every 2 s by a Coolsnap fx Photometrics (Roper Scientific) camera controlled by Metafluor software. After 100 s, particles were added to the cells (100 µl of a 250-µg/ml stock solution of particles) contained in phenol red-free RMPI 1640 medium. In some treatments, verapamil was also included with particles to give a final concentration of 100 µM. At 1,400 s, 10 µl of thapsigargin (10 µM; Sigma) were added, and a further series of images was captured until at least 1,800 s.

Preparation of nuclear extracts and EMSA. Rat alveolar macrophages were prepared and treated in 24-well plates as previously described for the UfCB dose-response and calcium inhibitor experiments. Nuclear extracts were prepared according to the method of Staal et al. (48). Cells were washed once with PBS and were incubated in 400 µl of buffer A containing 10 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.4 mM PMSF, 0.2 mM NaF, 0.2 mM NaVO3, and 1 µg/ml leupeptin on ice for 15 min. To lyse the cells, 25 µl of buffer B (10% Nonidet P-40) were added to each well, the cells were removed by scraping into Eppendorfs, and they vortexed for 15 s. Nuclei were collected by centrifugation at 900 g for 10 min, resuspended in 50 µl of buffer C (50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.4 mM PMSF, 10% glycerol, and 0.2 mM NaVO3), and agitated for 20 min at 4°C with vortex mixing every 5 min. Samples were centrifuged at 900 g for 10 min, the nuclear protein-rich supernatant was removed, and the protein concentration was measured using a Bio-Rad Protein kit. Ten micrograms of nuclear protein were incubated with 5x binding buffer (Promega, Southampton, UK) and {gamma}-32P-labeled NF-{kappa}B or AP-1 consensus oligonucleotide (Promega) according to the manufacturer's instructions. Two microliters (2.5 pmol) of unlabeled oligonucleotide were used as the cold competitor. Samples were electrophoresed using a 6% nondenaturing polyacrylamide gel, and DNA binding was assessed by phosphorimaging and quantified using densitometry with the UVP Grab and Gelplate program (Ultraviolet Products, Cambridge, UK).

Immunofluorescence of p50 and p65 in human monocytes. After the 4-h treatments, coverslips were washed twice with PBS and fixed with 3% formaldehyde in calcium/magnesium-free PBS for 20 min at room temperature. Coverslips were then washed three times with calcium/magnesium-free PBS, and excess aldehyde groups were quenched using 50 mM ammonium chloride for 10 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 4 min and washed three times with PBS, three times with 0.2% fish gelatin solution in PBS, and finally three times with PBS. All washes were over a 5-min period.

Coverslips were incubated for 1 h with rabbit polyclonal anti-NF-{kappa}B p50 or p65 subunit (Santa Cruz) diluted 1:200 in 0.2% fish gelatin solution and were washed three times with PBS, 0.2% fish gelatin, and PBS over 5 min for each wash. Cells were treated with a second antibody, FITC-labeled anti-rabbit IgG (SAPU Carluke), diluted 1:500 in 0.2% fish gelatin solution for 1 h at room temperature. Coverslips were mounted in Citifluor mounting medium (Agar Scientific) and allowed to dry before being viewed and analyzed. Images from each treatment were captured using a Photometrics (Roper Scientific) camera controlled by Metafluor software, and the intensity of the nuclear fluorescence was quantified using the same software. The intensity from the treatments was expressed as a percentage of the untreated cells.

Statistical analysis. Data from all of the experiments were analyzed using analysis of variance with Tukey's or Fisher's multiple comparison test. Significance was set at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular calcium concentration in particle-treated cells. Figure 1 shows the percentage change above control cells in intracellular calcium concentration in rat alveolar macrophages treated with particles. Data were collected after the addition of 200 µg/ml of either CB or UfCB, and the fluorescence intensity was measured at 340 and 380 nm and expressed as a ratio. Treatment of the macrophages with UfCB, but not CB, for 30 min induced a significant (P < 0.05) increase in intracellular calcium concentration compared with untreated cells (Fig. 1). The effect of UfCB treatment for 30 min on the cytosolic calcium concentration of rat alveolar macrophages was dose dependent (Fig. 2). Subsequent treatment with thapsigargin induced a further increase in cytosolic calcium, indicating that the cells remained viable. This response to thapsigargin also demonstrated a particle dose-dependent effect. Cotreatment of cells with particles and verapamil partially blocked the effect of particles alone on the intracellular calcium concentration since the level of calcium was not significantly different from control (Fig. 3).



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Fig. 1. Intracellular calcium concentration in rat alveolar macrophages during 30-min treatment with 200 µg/ml of carbon black (CB) or ultrafine carbon black (UfCB). Data represents the mean ± SE (n = 3) of the fura 2 fluorescence intensity ratio (340/380 nm) of cells expressed as a percentage above resting control cells. There was a significant difference (*P < 0.05) between untreated cells and UfCB-treated cells.

 


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Fig. 2. Intracellular calcium concentration in rat alveolar macrophages during 30-min treatment with 12.5, 25, or 50 µg/ml of UfCB. Data represent the mean ± SE (n = 3) of the fura 2 fluorescence ratio (340/380 nm) expressed as a percentage above resting control cells. There was a clear dose effect of UfCB particles on the intracellular calcium concentration.

 


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Fig. 3. Intracellular calcium concentration in rat alveolar macrophages after treatment with 200 µg/ml of UfCB with and without 100 µM verapamil (Verap). Data represent the mean ± SE (n = 3) of the fura 2 fluorescence ratio (340/380 nm) expressed as a % above resting control cells. There was a significant difference (*P < 0.05) between untreated cells and UfCB-treated cells but no significant difference between untreated cells and particle + verapamil-treated cells.

 

The alterations in intracellular calcium concentration induced by the UfCB treatment of individual cells occur with different kinetics. A video clip illustrating the cytosolic calcium concentration of individual cells over time can be observed in the accompanying video at http://146.176.1.22:8080/ramgen/caseweb/bio/av1.rm and http://146.176.1.22:8080/ramgen/caseweb/bio/av2.rm. This effect is also illustrated by examining images of the treated cells (Fig. 4). In untreated (particle-free) cells, the intense black color of the cells indicated a low, resting level of intracellular calcium. An increase in intracellular calcium (after treatment with UfCB particles) is indicated by a switch from black to blue/purple pseudocolor. It is clear that there is heterogeneity in the response to UfCB. UfCB produced a marked increase in intracellular calcium (Fig. 4), which was largely prevented by treatment with verapamil (Fig. 4).



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Fig. 4. Intracellular calcium concentration in rat alveolar macrophages after treatment with 200 µg/ml of UfCB shown as digitally enhanced images of fura 2-loaded rat alveolar macrophages. The pseudocolor image indicates the intracellular calcium concentration of the cells 700 s after treatment with medium (control), 200 µg/ml of UfCB, UfCB/verapamil, or verapamil (100 µM) alone. Black denotes low fluorescence ratio and hence calcium, whereas magenta indicates high calcium.

 

Transcription factor activation: rat alveolar macrophages. The electrophoretic mobility shift assay was used to investigate the effect of Uf particles on nuclear translocation of AP-1 and NF-{kappa}B in rat alveolar macrophages after particle treatment. Figure 5 demonstrates that the nuclear binding of AP-1 was almost doubled in UfCB-treated macrophages compared with untreated cells and CB-treated cells. Concomitant treatment with verapamil and UfCB particles reduced AP-1 binding back to control levels (Fig. 5), an effect that was significantly different from particles alone. Typical AP-1 gel shifts of these data are also shown in Fig. 5. We were unable to show a clear trend in NF-{kappa}B activation by any treatment using rat alveolar macrophages (data not shown), so we used human monocytes as targets.



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Fig. 5. 32P intensity of activator protein-1 (AP-1) transcription factor DNA binding in rat alveolar macrophage nuclear extracts after 4-h treatment with 200 µg/ml of CB, UfCB, UfCB/verapamil (UfCB+V), Verapamil (V; 100 µM), and LPS (1 µg/ml). Data represent the mean ± SE (n = 3) of the intensity as a percentage of the untreated control. The positive control consisted of HeLa cell nuclear extract. There was a significant difference (*P < 0.05) between UfCB and UfCB+Verapamil treatments. There was no significant difference between control and UfCB treatments.

 

Transcription factor activation: human monocytes. Human monocytes were used to investigate the activation of NF-{kappa}B by UfCB using a fluorescent staining technique. Figure 6 shows the fluorescence intensity in the nucleus of human monocytes stained with an antibody to the p65 (photographs) and p50 subunit of NF-{kappa}B after treatment with UfCB and calcium antagonists. A dose of 100 µg/ml of particles was chosen for this experiment to minimize interference in fluorescence observation and also because this concentration of particles was adequate to stimulate TNF-{alpha} protein release. The data are expressed as a percentage of the control (untreated) cells. There was a 30% increase (P > 0.05) in the intensity of p50 staining after treatment with UfCB compared with untreated cells. In addition, all calcium antagonists reduced the p50 staining intensity to control levels. The calmodulin inhibitor W-7, in the presence of UfCB, reduced the level to 60% of the control cells. The intensity of p65 staining after UfCB treatment was 68% greater than control cells. All the calcium antagonists reduced the staining intensity compared with particle treatment alone, but these differences were not statistically significant.



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Fig. 6. Immunofluorescence of p50 and p65 (photographs) subunits of NF-{kappa}B in human monocytes exposed to 100 µg/ml of UfCB for 4 h alone and in the presence of verapamil (100 µM), W-7 (250 µM), Trolox (25 µM), Nacystelin (NAL; 5 mM), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAM; 50 µM). Data represent the mean ± SE of the fluorescence intensity of the nuclei expressed as a percentage of control (untreated) cells.

 

TNF-{alpha} mRNA expression and release. Treatment of rat alveolar macrophages with a range of doses of UfCB for 4 h stimulated a concentration-dependent increase in the secretion of the proinflammatory cytokine protein TNF-{alpha} (Fig. 7A). At all concentrations of UfCB, there was a significant increase in TNF-{alpha} protein release compared with the control (P < 0.05). These increases in TNF-{alpha} protein were also reflected in a concentration-dependent increase in the TNF-{alpha} mRNA content at 4 h (Fig. 7B). The LPS-positive control increased TNF-{alpha} mRNA release to 95% of the GAPDH housekeeping gene. At a concentration of 200 µg/ml of UfCB, there was an approximate fourfold increase in the TNF-{alpha} mRNA of macrophages compared with the 100-µg/ml treatment, which was significantly greater than the control (P < 0.05). No TNF-{alpha} protein was detectable in supernatants before the 4-h time point.



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Fig. 7. Effect of treatment of rat alveolar macrophages for 4 h with UfCB on TNF-{alpha} protein (A) and TNF-{alpha} mRNA expression (B). Data represent the mean ± SE TNF-{alpha} (pg/ml) protein content in supernatants or the % mean ± SE of the gel band intensity expressed as a percentage of the GAPDH control obtained from 3 separate experiments. There was a significant difference (*P < 0.05) between untreated cells and cells treated with UfCB at all concentrations with respect to TNF-{alpha} protein and at 200 µg/ml with respect to mRNA. LPS treatment stimulated TNF-{alpha} mRNA, which was 95% of the GAPDH.

 

In contrast to the effects of UfCB on TNF-{alpha} mRNA and protein release, CB (200 µg/ml) had no significant effect on either of these parameters (Figs. 8 and 9). Coincubation of rat alveolar macrophages and UfCB (200 µg/ml) with either the calcium channel blocker verapamil (100 µM) or the calcium chelator BAPTA-AM (50 µM) significantly prevented the UfCB-induced enhancement of TNF-{alpha} protein release (Fig. 8A). In addition, verapamil, but not BAPTA-AM, significantly (P < 0.05) inhibited the UfCB-stimulated TNF-{alpha} mRNA production in the rat alveolar macrophages (Fig. 8B).



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Fig. 8. TNF-{alpha} protein release (A) and TNF-{alpha} mRNA expression (B) in rat alveolar macrophages treated with UfCB (4 h, 200 µg/ml) and the effect of the calcium channel blocker verapamil (100 µM) and the calcium chelator BAPTA-AM (50 µM). Data represent the mean ± SE (n = 3) of TNF-{alpha} protein release (pg/ml) and the % TNF-{alpha} mRNA of the GAPDH control. There was a significant difference between untreated cells and UfCB particle-treated cells (*P < 0.05) but no significant difference between untreated cells and particle-treated cells + verapamil. Particles + BAPTA-AM produced an effect similar to particles alone.

 


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Fig. 9. TNF-{alpha} protein release (A) and TNF-{alpha} mRNA expression (B) by rat alveolar macrophages treated with UfCB (4 h, 200 µg/ml) and the antioxidant NAL (200 µM). Data represent the mean ± SE of TNF-{alpha} (pg/ml) in supernatants and the % TNF-{alpha} mRNA expression of the GAPDH control from 3 separate experiments. There was a significant difference (*P < 0.05) between CB and UfCB particle-treated cells. Particles + NAL produced an effect similar to particles alone.

 

The antioxidant Nacystelin (5 mM) significantly inhibited the UfCB-induced increase in TNF-{alpha} protein release (P < 0.05; Fig. 9A); however, at this dose, Nacystelin did not prevent the UfCB-induced increase in TNF-{alpha} mRNA production (Fig. 9B). The inhibitors verapamil, BAPTA-AM, and Nacystelin did not alter the level of TNF-{alpha} mRNA or protein expression when added alone.

The inhibition of UfCB-induced TNF-{alpha} protein release by antagonists and antioxidants in rat alveolar macrophages was complemented by the data obtained for primary human monocytes (Fig. 10). First, treatment of the primary human monocytes with UfCB at the same dose and time used in the rat macrophage experiments stimulated a significant increase in TNF-{alpha} protein release (P < 0.05). Again, verapamil (100 µM) and BAPTA-AM (50 µM) were both able to inhibit this effect. Furthermore, the calmodulin inhibitor W-7 (25 µM) and the lipophilic antioxidant Trolox (50 µM) were also found to inhibit TNF-{alpha} protein release in response to UfCB exposure (Fig. 10).



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Fig. 10. TNF-{alpha} protein release by primary human monocytes treated with UfCB (4 h, 100 µg/ml) and the effect of the calcium channel blocker verapamil (100 µM), the calcium chelator BAPTA-AM (50 µM), the calmodulin inhibitor W-7 (250 µM), and the antioxidant Trolox (25 µM). Data represent the mean ± SE of TNF-{alpha} (pg/ml) in supernatants obtained from 4 separate experiments. There was a significant difference (*P < 0.05) between control and UfCB treatments.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased morbidity and mortality from respiratory and cardiovascular causes have been linked to increases in particulate air pollution, of which PM10 is a fraction (2, 46), and proinflammatory effects of PM10 very likely play a role (9, 11). Uf particles comprise the dominant number of particles in PM10, and these may play an important role in mediating the adverse health effects of PM10 (29). Inflammation is controlled by a complex series of intracellular and extracellular signaling events whose function is to control the production and secretion of extracellular signaling proteins, such as cytokines. This study aimed to investigate the effect of UfCB particles, as a surrogate for the Uf component of PM10, on oxidative and calcium-related cytokine regulation in rat alveolar macrophages and human monocytes.

Alveolar macrophages were used in this study because of their central role in dealing with deposited particles and their involvement in particle-mediated pulmonary inflammation (12). In our previous studies (4) using Uf polystyrene particles, we demonstrated a relationship between particle dose expressed as surface area and neutrophil recruitment into the lungs of particle-instilled rats. This important finding suggests a key role for surface area and may be an important consideration for the changes in calcium homeostasis, cytokine gene expression, and cytokine release observed in the present study.

In studies by Stone et al. (49) using a monocytic cell line (MM6), UfCB was found to enhance calcium influx into the suspended cells both at rest and after thapsigargin-mediated release of intracellular calcium stores. The present study confirms, by calcium imaging, that UfCB particles stimulate the entry of extracellular calcium into adhered primary rat alveolar macrophages on an individual cell basis. Imaging reveals that in these adhered primary cells, different macrophages respond at slightly different times after particle exposure and to differing degrees, either due to inherent differences between individual primary cells within the population or because of interaction with varying particle numbers as a consequence of heterogeneous particle dispersion within the culture system. Increases in cytosolic calcium can be a result either of cell signaling events or of cell death. To ensure that the cells remained viable after particle treatment, they were subsequently treated with thapsigargin. The cells, in all cases, continued to respond to thapsigargin, confirming their viable status.

Transcription factor activation and expression of proinflammatory genes such as TNF-{alpha} are key events in the initiation and control of inflammation. We demonstrate here that UfCB particles increase the DNA binding of the transcription factor AP-1 in rat alveolar macrophages and p50 and p65 nuclear localization in the human monocytes, an effect previously demonstrated with PM10 particles (25) and other pathogenic mineral dusts and fibers (45). The activation of proinflammatory transcription factors, such as nuclear factor of activated T cells, has been shown to be linked to calcium release from the endoplasmic reticulum stores (8); the same study postulates a role for calcium in the control of NF-{kappa}B and AP-1. In the present study, calcium was demonstrated to be involved in the activation and nuclear translocation of NF-{kappa}B subunits p50, p65, and AP-1 by UfCB since the calcium channel blocker verapamil inhibited NF-{kappa}B nuclear staining in the human monocytes and AP-1 DNA binding in extracts of the rat alveolar macrophages. Studies conducted by Santana et al. (43) and Jefferson et al. (24) have previously indicated a role for calcium in the control of these transcription factors. Furthermore, the calmodulin inhibitor W-7 inhibited the NF-{kappa}B nuclear localization, suggesting a role for this calcium-binding or sensory protein. Further investigation of different NF-{kappa}B subunits and inhibitors of NF-{kappa}B, such as I{kappa}B, are required to elucidate the effects of particles on signaling via these transcription factors. Such studies should address the kinetics of such responses and how they impact on modulation of cytokine expression.

The activation of NF-{kappa}B occurs by signaling pathways involving both calcium and ROS (1, 35, 40). Uf particles have been shown to generate ROS and cause oxidative stress (50). For example, treatment of the human type II epithelial cell line A549 with UfCB results in oxidative stress, as indicated by glutathione depletion (50), an effect also shown in J774 mouse macrophages (54). Furthermore, various Uf particles have been demonstrated to generate more free radicals and ROS than fine particles (50, 55), and the addition of transition metals and the large surface area of Uf particles may lead to potentiation of oxidant production and subsequent inflammation (54). It appears that these ROS are important in initiating or propagating the calcium signals in macrophages, since pretreatment of the cells with the antioxidants Nacystelin and mannitol diminished the calcium signaling events in MM6 cells induced by the UfCB treatment (51). We confirm here that Nacystelin also inhibits UfCB-induced nuclear translocation of NF-{kappa}B in the human monocytes, clearly implicating ROS in the molecular intracellular signaling response of macrophages via this transcription factor in response to Uf particles.

In addition to providing evidence that Uf particles stimulate the generation of ROS, calcium signaling events, and transcription factor activation, this study clearly demonstrates that UfCB stimulates both TNF-{alpha} mRNA production and protein release from rat and human macrophages. In these experiments, the calcium channel blocker verapamil, the intracellular calcium chelator BAPTA-AM, and the calmodulin inhibitor W-7 all significantly inhibited the production of TNF-{alpha} protein release by UfCB-treated macrophages. Again, this is strong evidence that calcium is involved in the regulation of this cytokine. In accordance with the experiments in which antioxidants inhibited both the intracellular calcium signal (50) and the activation of transcription factors, Nacystelin and the water-soluble vitamin E analog Trolox both prevented the activation of TNF-{alpha} protein release in response to UfCB exposure. This too is strong evidence that ROS are involved in the regulation of TNF-{alpha} protein release.

However, it is not clear from this study whether the oxidative pathways driving transcription factor activation and TNF-{alpha} protein release are due to ROS derived directly from the particles or from cell-generated ROS. Evidence from our previous studies suggests that ROS are partly responsible for driving the Uf particle-induced calcium response in macrophages, as this response was inhibited by antioxidants (50). The ability of oxidative stress to enhance cell signaling events via inositol 1,4,5-bisphosphate production and calcium signaling events has been demonstrated in various cell types, including endothelial cells (20) and macrophages (42). However, the relationship between intracellular calcium and oxidative stress may be complex, since oxidative stress has been shown to initiate changes in cytosolic calcium concentrations in cells that subsequently activate further production of ROS (31). Furthermore, these ROS may then further enhance calcium signaling (16), which would consequently enhance downstream events such as cytokine gene expression. One mechanism by which oxidative stress can alter calcium signaling is by oxidation of the calcium pumps in the endoplasmic reticulum (21, 52). Oxidation of these pumps would lead to depletion of the endoplasmic reticulum calcium store and hence disrupted calcium signaling.

It was noted that BAPTA-AM and Nacystelin were unable to prevent the production of TNF-{alpha} mRNA by the rat alveolar macrophages despite preventing the protein release. This suggests that calcium and ROS may also regulate the expression of such cytokines by posttranslational mechanisms, and the posttranslational regulation of TNF-{alpha} protein release has been previously published (26).

The control of TNF-{alpha} and protein release has been demonstrated to involve calcium in a number of circumstances, including during exposure of rat peritoneal macrophages to acetylated low-density lipoprotein (36) and by macrophages treated with endotoxin (6, 28). Alteration in TNF-{alpha} protein release is not always at the level of transcription, since some treatments can alter mRNA stability (22, 30, 32). It is clear from the literature that calcium is involved in both upregulation of TNF-{alpha} mRNA and protein release, but its role in posttranscriptional regulation and mRNA stabilization is unclear.

Recent studies (unpublished data) suggest that treatment of epithelial cells for just 30 min can stimulate increases in the mRNA content of cytokines such as IL-8, suggesting that the particles may enhance RNA stability instead of stimulating synthesis de novo. Studies are ongoing to investigate this observation further.

We have previously demonstrated that UfCB produces its increased inflammatory effects via mechanisms other than the leaching of soluble components from the particle surface (5). Transition metals are an important source of free radicals, which are important in PM10-stimulated lung inflammation (29). The treatment of PM10 particles with transition metal chelators diminishes the proinflammatory effects of PM10 (19). Fine CB had less activity in all of the assays used here than a similar mass of UfCB. The increase in TNF-{alpha} protein release induced by UfCB exposure of rat alveolar macrophages was dose dependent. There was a clear relationship between either the mass or surface area of the particle treatment and the TNF-{alpha} protein released (r2 = 0.9461). As in previous similar studies (5), in the present study, we do not consider soluble components of the particles to be important in the calcium events shown here. Instead, surface area and ROS generation are likely to be key factors.

The present study has shown that Uf particles may exert their increased proinflammatory effects, at least in part, by modulating intracellular calcium. Activation of transcription factors, such as AP-1 and NF-{kappa}B, through a calcium and ROS-mediated mechanism may cause inflammation and contribute to the adverse health effects induced by Uf particle exposure (37). The role of calcium and ROS in other cellular and molecular responses to Uf particle and PM10 exposure are under investigation.


    ACKNOWLEDGMENTS
 
Present addresses: M. Dehnhardt, Molecular Neuroimaging Group, Research Centre Juelich, Institute for Medicine, Leo Brandt Str., 52425 Juelich, Germany; P. Gilmour, Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina, Chapel Hill, NC 27599.

GRANTS

This study was generously funded by the Colt Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Brown, School of Life Sciences, Napier Univ., 10 Colinton Rd., Edinburgh EH10 5DT, United Kingdom (E-mail: da.brown{at}napier.ac.uk).

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.


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