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Published ahead of print on June 10, 2004, doi:10.1165/rcmb.2003-0300OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 292-301, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0300OC

Inflammatory Time Course after Quartz Instillation

Role of Tumor Necrosis Factor-{alpha} and Particle Surface

Catrin Albrecht, Roel P. F. Schins, Doris Höhr, Andrea Becker, Tingming Shi, Ad M. Knaapen and Paul J. A. Borm

Institut für Umweltmedizinische Forschung (IUF), Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany

Address correspondence to: Dr. Catrin Albrecht, Institut für Umweltmedizinische Forschung (IUF) gGmbH, Heinrich-Heine-University Düsseldorf, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany. E-mail: catrin.albrecht{at}uni-duesseldorf.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammation has been suggested as the key factor in the development of quartz-induced fibrosis and carcinogenesis, and particle surface properties are argued as an important characteristic responsible for these pathologic alterations. To evaluate the effect of surface modification on acute and subchronic inflammation, female Wistar rats were intratracheally instilled with 2 mg native quartz, or quartz coated either with polyvinyl-pyridine-N-oxide or with aluminium lactate. Various markers of lung toxicity, inflammation, and oxidative stress were found to be enhanced at 3, 7, 21, and 90 d after instillation of native quartz. Quartz-treated animals also showed enhanced immunostaining of nuclear factor-{kappa}B (NF-{kappa}B) in alveolar macrophages and lung epithelium, as well as reduced I{kappa}B{alpha} levels in whole lung homogenate. Both surface modifications were found to inhibit most of the effects as observed with native quartz. NF-{kappa}B activation was also observed in vitro in rat lung epithelial cells following treatment with lavage fluid from quartz-treated animals, as well as with conditioned medium of quartz-treated macrophages, and these effects appeared to be at least partly tumor necrosis factor-{alpha}–independent. In conclusion, the persistent subchronic inflammatory lung response after quartz exposure appears to be particle surface–driven and is associated with NF-{kappa}B activation in both alveolar macrophages and the lung epithelium.

Abbreviations: aluminum lactate, AL • bronchoalveolar lavage fluid, BALF • 5,5-dimethyl-1-pyrroline-N-oxide, DMPO • fetal calf serum, FCS • interleukin, IL • macrophage inflammatory protein-2, MIP-2 • myeloperoxidase, MPO • nuclear factor-{kappa}B, NF-{kappa}B • superoxide anion, O2- • hydroxyl radicals, ·OH • phosphate-buffered saline, PBS • polyvinyl-pyridine-N-oxide, PVNO • rat lung epithelial cells, RLE cells • reactive nitrogen species, RNS • reactive oxygen species, ROS • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Respirable quartz has been classified as a human lung carcinogen based on the findings in human epidemiologic studies and experimental studies (1). However, mechanisms involved in quartz-induced carcinogenesis as well as fibrosis remain largely unclear. Previous studies have indicated that the inflammatory reaction in the lung triggered by quartz is a crucial step in the development of both lung cancer and fibrosis (2–4). Inflammation, as well as the particle surface itself, are well known to generate several reactive oxygen as well as nitrogen species (ROS/RNS), such as hydroxyl radicals (•OH) and superoxide anion (O2-) (5, 6). The connection between ROS and silica-induced carcinogenesis was reviewed by Shi and coworkers (7).

Few studies are available that report on the time course of inflammation up to the moment where collagen formation begins and fibrosis develops. Porter and colleagues (8) have recently demonstrated, using an inhalation model, that both inflammation and fibrosis showed a slow initial increase followed by an exponential rise after 40 d at a time point where the silica burden was increased over 3 mg/lung. This suggests that on the one hand a certain dose is necessary to initiate a persistent inflammatory response and on the other hand that acellular components at a certain level are necessary to drive the lung response.

An important key factor in the inflammatory cellular response is the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) which controls the expression of several genes which have multiple functions in the inflammatory processes (9). Synergism of ROS/RNS and tumor necrosis factor (TNF)-{alpha} in the induction of NF-{kappa}B activation in rat lung epithelial cells has been shown by Janssen-Heininger and coworkers (10). Furthermore, Driscoll and colleagues (11) demonstrated the role of epithelial cells in macrophage inflammatory protein (MIP)-2 production and NF-{kappa}B activation by direct quartz exposure. Inhibition by antioxidant treatment suggests a role for ROS in particle elicited NF-{kappa}B activation (12), e.g., as shown with antioxidants including TMTU (11) and curcumin (13). Lentsch and coworkers (14) demonstrated the crucial role of macrophages for pulmonary NF-{kappa}B activation and onset of inflammation. This further strengthens the reason to study bronchoalveolar lavage fluid (BALF) effects on NF-{kappa}B activation in lung epithelial cells, rather than looking at the direct effect of quartz on NF-{kappa}B activation as previously demonstrated by Driscoll and colleagues (11). Therefore, in this study, special attention was placed on the mechanism of NF-{kappa}B activation to characterize the ongoing inflammatory process in the lung. The release of proinflammatory mediators from particle-exposed macrophages may be important for stimulation of cytokine release from lung epithelial cells, thus amplifying the inflammatory response. As a model for interaction between macrophages and epithelial cells, Jimenez and coworkers (15) treated human lung epithelial cells with conditioned media from monocyte-derived macrophages stimulated with PM10 or fine as well as ultrafine titanium dioxide. Only PM10 treatment caused an increase in NF-{kappa}B and activator protein-1 DNA binding and enhanced interleukin (IL)-8 mRNA levels as well as transactivation of IL-8. Analysis of these conditioned media revealed a marked increase in tumor necrosis factor-{alpha} (TNF-{alpha}) protein levels and an enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-{alpha}–neutralizing antibodies significantly reduced IL-8 production.

Modification of the particle surface by coating with polyvinyl-pyridine-N-oxide (PVNO) or aluminium lactate (AL) is a powerful tool to influence biological effects of particles and has been demonstrated in several studies. Interactions between the quartz surfaces and aluminum ions (e.g., those present in kaolinites and bentonites) are considered to be a common occurrence in nature, and have been suggested to provide some explanation for the observed variability in adverse health effects of quartz in different industries (1, 5, 16). Indeed, different workplace quartzes have been shown to contrast in their inflammogenic potency in rat lungs, and addition of clay-based compounds during commercial grinding of quartz has been forwarded as a tool to reduce its biological activity (6, 17, 18). Our group has shown previously that coating with either PVNO or AL inhibits hydroxyl radical generation of the quartz particles, oxidative DNA damage in epithelial cells in vitro (19), in vitro toxicity in rat lung epithelial (RLE) cells and neutrophilic burst (20). Furthermore, we demonstrated that coating of DQ12 with PVNO or AL reduced acute inflammation (21), expression of NF-{kappa}B and MIP-2 (22) and strand breaks in isolated lung epithelial cells 3 d after quartz instillation (20).

In the present study, we have hypothesized that the inhibition of subacute inflammation, if sustained, may reduce the onset and extent of epithelial hyperplasia as preneoplastic lesion as well as fibrosis. The purpose of this study was therefore to evaluate the inflammatory response elicited by different surface modified quartzes up to 90 d after instillation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The animals were housed and maintained in an accredited on-site testing facility, responding to the guidelines of the Society for Laboratory Animals Science (GV-SOLAS). Eight-week-old female Wistar rats (220 g; Janvier, Le Genest St. Isle, France) were used for this study. All animals were allowed food and water ad libitum. The animals were housed on hardwood bedding in plastic cages in an air-conditioned animal room (23 ± 2°C) with a regular 12 h light/dark cycle under SPF conditions.

Particle Characteristics and Coating Procedure
Dörentruper quartz (DQ12) was used as native quartz as well as for the preparation of surface-modified quartz. DQ12 was baked at 220°C for 16 h to inactivate possible endotoxin on particle surface. The coating procedure was performed as described previously (19). Briefly, quartz was suspended at a concentration of 5 mg/ml in 1% solutions of either PVNO or AL in distilled water. Samples were subsequently sonicated for 5 min, and agitated for 5 h at room temperature. The native quartz was suspended in a concentration of 5 mg/ml in distilled water, sonicated and agitated for the same time intervals. After agitation, each suspension was washed three times by centrifugation (5 min, 13,000 rpm) in distilled water. One batch of each modified quartz was prepared for the whole experiment to avoid possible variability in coating efficiency and then aliquoted, air dried, and stored in the dark. Immediately before intratracheal instillation, aliquots of the dried dusts were resuspended in 1 ml phosphate-buffered saline (PBS), resulting in a concentration of 5 mg/ml, sonicated (5 min), and stirred until application. Investigation by electron microscopy shows that the coating procedure did not affect the particle size distribution or aggregation (Figure 1A).



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  		Figure 1. (A) Distribution frequency of particle size of native (squares) and surface modified (triangles, DQ12-PVNO; circles, DQ12-AL) quartzes were determined by electron microscopy before intratracheal instillation. (B) Hydroxyl radical generation of native and surface modified quartz DQ12 particles were measured by ESR. Shaded bars, t = 0; hatched bars, 1 yr later.

 
The efficacy of the coating procedure was investigated for PVNO by spectrophotometry and for AL by Atomic Absorption Spectrometry (AAS) as described elsewhere (19). The adsorbed amounts on the quartz preparations used for the present study were found to be 11 µg PVNO/mg quartz and 1.6 µg aluminum/mg quartz.

The ability of the different quartz preparations to generate hydroxyl radicals (·OH) was measured by electron spin resonance technique (ESR) immediately before the first instillation time point as well as before the last instillation time point nearly 1 yr later. The particles were suspended in distilled water, sonicated for 5 min, and this particle suspension (final concentration 10 mg/ml) mixed with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 25 mM) and hydrogen peroxide (125 mM). The mixture was incubated in the dark and shaken continuously at 37°C for 15 min, and subsequently filtered through a 0.2-µm filter (Minisart RC15; Sartorius AG, Göttingen, Germany). The clear filtrate was transferred to a 100-µl glass capillary and the ·OH generation measured in a Miniscope ESR spectrometer (Magnetech, Berlin, Germany). A mixture of water, H2O2, and DMPO was used as a negative control. The ESR spectra were recorded at room temperature using the following instrumental conditions: Magnetic field: 3,360 G; sweep with: 100 G; scan time: 30 s; number of scans: 3; modulation amplitude: 1,800 G. Quantification was done on first derivation of EPR signal of DMPO-OH quartet as the sum of total amplitudes, and outcomes are expressed as the total amplitude in arbitrary units (A.U.). Measurement of •OH generation by ESR was used to assess the stability of the instillation samples. Surface coating with PVNO as well as with AL showed a reduced DMPO-OH formation. The relation between the three quartz preparations were consistent over the entire experimental period (~ 1 yr) as shown by two independent experiments before instillation of the first and last experimental groups (Figure 1B).

Animal Treatment
After anesthetization (Isofluran; Essex Pharma GmbH, Munich, Germany), animals were intratracheally instilled (400 µl) with PBS as vehicle control, 2 mg DQ12 quartz or DQ12 (2 mg) coated with either PVNO (DQ12+PVNO) or with AL (DQ12+AL). As additional controls animal groups received the coating substances alone (i.e., 22 µg PVNO or 35 µg AL, representing 1.6 µg aluminum) based on the coating efficiencies as observed for the quartz preparations. At Days 3, 7, 28, and 90 after instillation, animals were killed by deep anesthetization with pentobarbital (50 mg/kg body weight) followed by exsanguination via the A. abdominalis.

Bronchoalveolar Lavage
Lungs of five animals per treatment group and time point were lavaged as described previously (23). Briefly, lungs were infused four times with 5 ml PBS and drained by gravity. The total recovery of the BALF was 90–95% of the instilled PBS volume (range: 18–19 ml). The lavage fluid was spun (500 x g, 10 min, 4°C) and cells were collected for cell count and differentiation. Supernatants were spun again (900 x g, 10 min, 4°C) and investigated for parameters of cell toxicity, inflammation, and oxidative stress as summarized in Table 1.


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  		TABLE 1. Parameters investigated in the BAL to determine cell toxicity, inflammation, cell activation, and oxidative stress

 
Lung Fixation and NF-{kappa}B Immunohistochemistry
Lungs of five additional animals per treatment group and time point were instilled in situ with 4% paraformaldehyde/PBS (pH 7.4) under atmospheric pressure, removed, fixed in the same solution for at least 12 h, dehydrated, and paraffin embedded. After deparaffinization of 3-µm lung sections with Xylene (2 x for 10 min), slides were washed successively for 10 min in acetone, acetone:TBS (1:1), and TBS. This was followed by inactivation of endogenous peroxidase with 0.3% hydrogen peroxide for 30 min. After washing with TBS, sections were permeabilized in citrate buffer (pH 6.0) by microwave treatment followed by washing with TBS. To avoid nonspecific binding, the sections were blocked with goat serum (1:65) for 1 h. The sections were then incubated with a polyclonal primary antibody against p65 of NF-{kappa}B (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was performed by incubation with a secondary biotinylated goat-anti rabbit antibody (1:200; Vector Laboratories, Burlingame, CA) followed by the streptavidin-biotin system (Vectastain Elite Kit; Vector Laboratories) according to the manufacturer's protocol. Diaminobenzidine (Sigma, Deisenhofen, Germany) was used as a substrate, and the slides were counter stained with hematoxylin. After washing with distilled water, slides were dehydrated and covered in DePex (Serva, Heidelberg, Germany). For the negative control, serial sections were incubated with rabbit IgG (Vector Laboratories) instead of the primary antibody at the same concentration.

Western Blotting of I{kappa}B{alpha}
Lung tissue was removed from the animals, chopped into small pieces, aliquots were snap frozen in liquid nitrogen and stored at –80°C. For preparation of whole protein, lung tissue from different treatments and time points was homogenized with lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate in PBS) containing freshly added protease inhibitors (Complete cocktail; Roche, Penzberg, Germany). Homogenate-lysis buffer-mix was incubated for 30 min on ice and spun at 15.000 x g for 20 min at 4°C. Protein concentrations were determined by Bio-Rad (Munich, Germany) Assay (according to the Bradford method). Samples were analyzed by Western blotting as described previously (26). Briefly, samples were electrophoresed at equal protein concentrations (20 µg) in 10% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto Nitrocellulose membranes (Schleicher and Schuell). Nonspecific protein binding was blocked with 5% dried milk powder and 0.05% Tween-20 in PBS. Immunolocalization of I{kappa}B{alpha} protein was performed using polyclonal I{kappa}B{alpha} rabbit IgG (C-21, 1:1,000; Santa Cruz Biotechnology Inc.) and anti-rabbit-IgG whole protein horseradish peroxidase conjugated (1:3,000; Sigma). Band formation was visualized using the ECL-reagent/detection system (Amersham Bioscience Europe, Freiberg, Germany). Quantification was performed by computer-assisted densitometry scanning using a documentation system (Bio-Rad) with associated software (geldoc system). For each time point, samples from four animals per treatment group were quantitated.

Treatment of Rat Lung Macrophages
The rat lung macrophage cell line NR8383 was used as a model to investigate macrophage–epithelial cell interactions. Macrophages were seeded in 60-mm culture dishes and cultivated in Ham's F 12K medium/15% fetal calf serum (FCS)/1% penicillin/1% streptomycin/1% glutamine. At 60% confluence, cells were treated with 40 µg/cm2 DQ12, or surface modified DQ12 as described under 2.2. Controls received PBS only. After 24 h supernatants were spun to remove nonadherent cells and to avoid cell damage at 1,350 rpm for 5 min. Supernatants were then spun again at 13,500 rpm for 5 min to remove any cell debris and particles. Supernatant was used to condition medium for treatment of RLE cells to determine the interaction between alveolar macrophages and epithelial cells. Enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Wiesbaden, Germany) was used to determine the TNF-{alpha} values of NR supernatants so as to calculate the appropriate concentration of anti–TNF-{alpha} antibody to be used (R&D Systems) for TNF-{alpha} neutralization in the supernatant according to manufacturer's protocol.

Treatment of RLE Cell Line
Immortalized RLE cells (27) were seeded in four chamber culture slides (BD Falcon, Heidelberg, Germany) and cultivated in Ham's F 12 medium/5% FCS/1% penicillin/1% streptomycin/1% glutamine. At 90% confluence, medium was discarded and cells were cultured in medium with 0.1% FCS for 18 h. Cells were then treated with rat TNF-{alpha} (R&D Systems) at concentrations of 0.1, 1, or 10 ng/ml to establish the immunohistochemical method for detection of NF-{kappa}B activation. For further experiments RLE media were conditioned either with BALF of animals of the different treatment groups as described under 2.3. (100 µl + 250 µl medium/well) or with supernatant of quartz treated macrophages (100 µl + 250 µl medium/well) for 2 h at 37°C. Immunohistochemistry was performed as described earlier in the methods section using Vector SG (Vector Laboratories) as a substrate and Nuclear Fast Red (Vector Laboratories) as nuclear counterstaining. The strong contrast allows a clear distinction between negative red and positive gray nuclei. Eight hundred cells per treatment were randomly counted under a microscope by an independent investigator (magnification x400; Leitz Meßtechnik GmbH, Wetzlar, Germany) whereby all cells with a more intensive gray stained nucleus compared with the plasma, indicative of nuclear translocation of NF{kappa}B, were determined as positive.

Statistical Analysis
Data from BAL analysis are presented as mean ± SD for n = 5 animals per treatment and time. Immunocytochemistry data for NF-{kappa}B in RLE cells represent mean ± SD for n = 3 independent experiments. Data from I{kappa}B{alpha} analysis are presented as mean ± SD for n = 4 animals. Results were statistically analyzed by ANOVA and post hoc testing (Tukey) unless stated otherwise.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DQ12 Induces an Acute and Subchronic Inflammatory and Oxidative Stress Response in the Rat Lung that Continues up to 90 D
Instillation of 2 mg DQ12 {alpha}-quartz resulted in an early increase in the total cell number in the BALF, which was further enhanced with time until the 90 d time point (Figure 2A). This increase is reflected by the total number of alveolar macrophages as well as neutrophils, the main cell fractions within the BALF, which were both increasing with time (data of cell differential not shown). As a characteristic of the inflammatory process, the percentage of neutrophils in the BALF appeared to increase up to 65% (Figure 2B). The cell influx after DQ12 instillation was found to be prevented by both coatings. PVNO coating of the quartz led to a reduction in the total cell number from 67% up to 93% compared with the native quartz treatment. Surface modification with AL reduced the total cell influx between 42 and 83%. Animal groups that were instilled with coating substances alone did not show any differences compared with PBS treated rats. This is also valid for all parameters described further on (data not shown). The neutrophilic inflammation appeared to be inhibited at the various time points upon coating of the quartz particles with PVNO (i.e., 76–99%) and AL (i.e., 48–86%). For PVNO, the reduction in the percentage of PMN was significant after 7, 21, and 90 d (90–63%), whereas for AL the PMN percentage was significantly reduced only at Day 7 (43%).



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  		Figure 2. Inflammation and cytotoxicity were determined by measurement of total cells (A), percentage of PMNs (B), total protein (C), LDH (D), and alkaline phosphatase (E) in the bronchoalveolar lavage 3, 7, 28, and 90 d after intratracheal instillation of native and surface-modified quartzes. Data are presented as mean ± SD (n = 5). Results were statistically analyzed by ANOVA and post hoc testing (Tukey). Significant differences of the particle instilled animals versus PBS controls are shown by ***P < 0.001, **P < 0.01, *P < 0.05. Significant differences of the surface-modified quartzes versus native quartz are shown by °°°P < 0.001, °°P < 0.01, °P < 0.05. Diamonds, PBS; squares, DQ12; triangles, DQ12-PVNO; circles, DQ12-AL.

 
Total protein in the BALF was significantly increased at Days 28 and 90 after instillation of native quartz, as shown in Figure 2C. At these time points, the surface coatings reduced this effect (PVNO from 78 up to 82%, AL from 64 up to 53%).

A significant increase in lactate dehydrogenase (LDH) levels in BALF was observed for all time points after quartz instillation, as shown in Figure 2D, whereas a strong increase was observed between the 28 and 90 d time points. Both coatings inhibited the cytotoxicity of quartz, PVNO between 67 and 93%, and AL between 42 and 83%. The lower effect of AL coating is also demonstrated by an significant increase of LDH in this animal group compared with the control.

In contrast, alkaline phosphatase activity in the BALF showed only a significant increase at Day 7 after instillation with native quartz (Figure 2E), and this effect was reduced by both surface modifications (PVNO at 42% and AL at 31%).

ß-Glucuronidase was already increased as early as 3 d after native quartz instillation and remained significantly increased over the whole observation period (Figure 3A). This induction was inhibited by both surface modifications at all time points investigated. The effect of PVNO was between 88 and 93%, and the effect of AL was between 74 and 83%. Despite significant reduction of quartz effects by AL-coating, ß-glucuronidase values in the 90-d samples were significantly increased compared with the control values.



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  		Figure 3. ß-Glucuronidase (A) and MIP-2 (B) characterize the activation status of inflammatory cells in the rat lung after animal treatment with different quartzes. Data are presented as mean ± SD (n = 5). Results were statistically analyzed by ANOVA and post hoc testing (Tukey). Significant differences of the particle-instilled animals versus PBS controls are shown by ***P < 0.001; **P < 0.01; *P < 0.05. Significant differences of the surface-modified quartzes versus native quartz are shown by °°°P < 0.001; °°P < 0.01; °P < 0.05. Diamonds, PBS; squares, DQ12; triangles, DQ12-PVNO; circles, DQ12-AL.

 
MIP-2 levels in the BALF were also significantly increased over the entire experimental period (Figure 3B), whereas TNF-{alpha} levels remained under the detection limit (5 pg/ml, data not shown). Coating of the quartz surface by PVNO appeared to prevent the increase of MIP-2 up to 4 wk (21–51%), coating with AL until Day 28 (32–43%). At Day 90 PVNO indeed reduces the quartz caused MIP-2 increase significantly, nevertheless both coated quartz preparations showed a significant increase compared with the control values.

Myeloperoxidase (MPO) activity (Figure 4A) as well as Trolox equivalent antioxidant capacity (TEAC, Figure 4B) were significantly increased after native quartz instillation at all time points. Both coatings appeared to inhibit the MPO activity (PVNO: 92–100%; AL: 78–100%) as well as the TEAC values (PVNO: 51–70%, AL: 43–57%). However, after surface modification by AL a significant increase in the TEAC values were observed at Day 90.



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  		Figure 4. Myeloperoxidase (A) and TEAC measurements (B) characterizing the oxidative stress situation in the lung of the treated animals. Data are presented as mean ± SD (n = 5). Results were statistically analyzed by ANOVA and post hoc testing (Tukey). Significant differences of the particle instilled animals versus PBS controls are shown by ***P < 0.001; **P < 0.01; *P < 0.05. Significant differences of the surface-modified quartzes versus native quartz are shown by °°°P < 0.001; °°P < 0.01; °P < 0.05. Open bars, PBS; solid bars, DQ12; hatched bars, DQ12-PVNO; dotted bars, DQ12-AL.

 
Activation of the NF-{kappa}B Pathway in Rat Lungs upon Treatment with DQ12
Two independent approaches were used as an indicator of the activation of the proinflammatory transcription factor NF-{kappa}B in vivo, upon treatment with the different quartz preparations: (i) degradation of the cytosolic NF-{kappa}B inhibitor protein I{kappa}B{alpha} in whole lung homogenates, and (ii) immunohistochemistry in lung sections for the p65 subunit of NF-{kappa}B. Western blot analysis results of I{kappa}B{alpha} protein levels in rat lung homogenates are shown in Figure 5. Both 3 d (Figure 5A) and 90 d (Figure 5B) after instillation, the lungs of the quartz-treated animals tended to have lower I{kappa}B{alpha} levels, compared with the untreated animals. The difference was significant at the 90-d time point, and at this time point, also animals that received DQ12+AL showed a reduced amount of I{kappa}B{alpha} in whole lung protein compared with the control animals (see Figure 5B). The I{kappa}B{alpha} levels in the animals that were treated with DQ12+PVNO were similar to those observed in the control animals, irrespective of the time point.



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  		Figure 5. I{kappa}B{alpha} protein in whole lung homogenates from rats at 3 (A) or 90 d (B) after instillation of particles or PBS. The graphs represent the mean ± SD of the band densities of protein from four animals per treatment group. Results were statistically analyzed by ANOVA and post hoc testing (LSD). Significant differences of particle treatments versus PBS are shown by **P < 0.01; *P < 0.05. (The inserts show a representative blot for one animal per treatment group.)

 
Immunohistochemical analysis of NF-{kappa}B activity in lung sections from rats from different treatment groups is shown in Figure 6. NF-{kappa}B activation was detected in lung sections of native quartz–treated animals at all time points. As expected, staining was observed in alveolar macrophages, but also in bronchiolar as well as alveolar epithelial cells indicating an active participation of the rat lung epithelium in the inflammatory response to quartz in vivo. Representative pictures of immunohistochemistry are shown for the 3 d time point (Figures 6A–6D) and the 90 d time point (Figures 6E–6H). After quartz instillation the NF-{kappa}B signal appears to be increased at acute (Figure 6B) and subchronic (Figure 6F) time points compared with the controls (Figures 6A and 6E). Animals instilled with the surface modified quartzes show a reduced NF-{kappa}B signal as compared with the native quartz (DQ12-PVNO: Figures 6C and 6G, DQ12-AL: Figures 6D and 6H). Interestingly, at 90 d, the NF-{kappa}B staining tended to be higher for the animals that were treated with DQ12+AL compared with the animals receiving DQ12+PVNO, in line with our I{kappa}B{alpha} blotting observations (Figure 5B).



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  		Figure 6. Immunohistochemical staining for NF-{kappa}B p65 in lung sections taken from control animals (A, E), native DQ12 (B, F)–, DQ12-PVNO (C, G)–, or DQ12-AL (D, H)–treated animals at 3 (A–D) or 90 (E–H) d after a single intratracheal instillation. Original magnification: x1,000.

 
Role of Epithelial Cells in Inflammatory Response of the Rat Lung
To investigate the possible role of epithelial cells we established two complementary strategies to look for the nuclear translocation of NF-{kappa}B. First, we investigated NF-{kappa}B activation in RLE cells upon treatment with the BALF from different treatment groups. Furthermore, we conditioned RLE medium with supernatant of dust-treated macrophages to investigate signals from macrophages which could stimulate NF-{kappa}B activation in epithelial cells. TNF-{alpha} values in the BALF of treated animals and supernatants of macrophages were determined by ELISA. Whereas TNF-{alpha} values in the lavages were under the detection limit, i.e., < 5 pg/ml (data not shown), results of TNF-{alpha} release from dust-exposed macrophages are shown in Table 2. Treatment of macrophages with native DQ12 led to an increase in TNF-{alpha} production, which could be reduced by surface modification either with PVNO or with AL. Because TNF-{alpha} functions as a well known candidate for NF-{kappa}B activation, recombinant rat TNF-{alpha} was used as a positive control for the induction of NF-{kappa}B activation in lung epithelial cells. A dose-dependent increase of cells with positive nuclear staining was observed. Quantification of the frequency of positive cells from three independent experiments showed a significant dose dependent increase after treatment with 1 and 10 ng/ml TNF-{alpha} (data not shown).


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  		TABLE 2. Measurement of TNF-{alpha} (pg/ml) in the supernatant of quartz-treated macrophages

 
The effects of the treatment of RLE cells with BALF from animals from the 3 d time point are shown in Figure 7. Representative immunohistochemistry findings can be seen in Figure 7A, and quantification of NF-{kappa}B translocation in the RLE cells, using the BALF from three animals per treatment group are shown in Figure 7B. The BALF from quartz-treated rats (3 d) caused a significant enhancement of NF-{kappa}B translocation in the RLE cells compared with the BALF from the untreated animals. BALF from animals instilled with coated quartz particles did not induce a significant NF-{kappa}B translocation. In an in vitro model we endeavored to modulate the in vivo situation of macrophage–epithelial cell communication leading to NF-{kappa}B activation in epithelial cells. Results of the quantification of positive nuclei are shown in Figure 8A. Treatment of RLE cells with the supernatant from DQ12-treated macrophages caused an activation of NF-{kappa}B in the epithelial cells compared with treatment with the supernatant from PBS-treated macrophages. Surface modification of DQ12 either with PVNO or with AL inhibited the NF-{kappa}B translocation in RLE cells. Because TNF-{alpha} values in the BAL were below the detection limit, we hypothesized that TNF-{alpha} may not be a key player for NF-{kappa}B activation in our current study. Therefore, in a second experiment, supernatants from DQ12-treated macrophages were pre-incubated with the calculated amounts of anti–TNF-{alpha} neutralizing antibody. This did not reduce the NF-{kappa}B signal in three independent samples, indicating that constituents other than TNF-{alpha} are involved. Results of quantification are shown in Figure 8B.



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  		Figure 7. (A) Immunohistochemical staining for NF-{kappa}B p65 in RLE cells after treatment with bronchoalveolar lavage fluid of particle-exposed animals and PBS-treated controls. (B) Quantification of cells with positive-stained nuclei. Data are presented as mean ± SD (n = 3). Results were statistically analyzed by ANOVA and post hoc testing (Tukey). Significant differences of particle treatments versus PBS are shown by ***P < 0.001. Significant differences of the surface-modified quartzes versus native quartz are shown by °°°P < 0.001. Original magnification: x1,000.

 


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  		Figure 8. Quantification of NF-{kappa}B nuclear translocation in RLE cells after treatment with supernatant from macrophages treated with native and surface-modified quartzes (A) and after preincubation of supernatant of DQ12-reated macrophages with a neutralizing anti–TNF-{alpha} antibody (B). Data are presented as mean ± SD (n = 3). Results were statistically analyzed by ANOVA and post hoc testing (Tukey). Significant differences of particle treatments versus PBS are shown by ***P < 0.001; **P < 0.01. Significant differences of the surface-modified quartzes versus native quartz are shown by °°°P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Respirable quartz is well known to induce fibrosis and cancer in rats, and lung inflammation is considered as the key event in the induction of DNA damage, mutagenesis and cell proliferation. In the present study, by investigating the time course of lung inflammation after a single intratracheal instillation of quartz, we have shown that surface modification of quartz with either PVNO or AL leads to a significant inhibition of the induction as well as the persistence of lung inflammation. Our data also indicate that NF-{kappa}B activation in macrophages, as well as in epithelial cells, may be involved in this inflammatory process, but not necessarily driven by TNF-{alpha} production in the lung.

As chronic inflammation plays a major role in the development of lung tumors in rats, we chose a quartz dose which was known to induce a persistent inflammation. Driscoll and coworkers (2) investigated the lung toxicity response after intratracheal instillation of 5, 10, 50, and 100 mg/kg quartz (Min-U-Sil) or titanium dioxide in Fisher rats (~ 200 g). At all doses, quartz was found to induce an increase in total protein and LDH in the lavage fluid at 7, 14, and 28 d after instillation as well as the occurrence of fibrosis at later time points. In contrast, titanium dioxide, used as a low toxic particle control, showed toxicity (LDH, protein) over the whole observation period after instillation of 50 mg/kg, whereas at a dose of 10 mg/kg, toxicity was only transiently induced, i.e., at Day 7. We used a dose of 10 mg/kg, to induce a clear positive response by quartz during the study period regarding toxicity, inflammation and the induction of oxidative stress. Our results correspond to the study of Ernst and colleagues (28), which shows significant increases in the total number of leukocytes, the percentages of PMN and lymphocytes, as well as lung wet weight, upon instillation of the same dose as used in the present study. Compared with the subchronic inhalation study (15 mg/m3, 6 h/d, 5 d/wk, 116 d) performed by Porter and coworkers (8), which led to a lung burden of ~ 6 mg, here we used a quartz dose three times lower. This likely explains the relative absence of high toxicity for lung parenchyma cells, especially type II cells in our study. This is, for instance, indicated by the fact that alkaline phosphatase, as a marker for type II cell toxicity, was only transiently increased at Day 7 after instillation. Because our administered dose was only 2 mg, we assume that the occurrence of epithelial hyperplasia and fibrosis do not result from particle overload, high toxicity, and subsequent lung remodeling, but rather as a consequence of the chronic presence of inflammatory cells and mediators.

In the context of human occupational exposure the dose can be considered as moderate and would correspond to a lung burden of 800 mg. This particle load is achieved by a 1-yr exposure (8 h/d, 200 d/yr) to 1 mg/m3 respirable quartz, assuming 50% deposition fraction. Although this may seem high in relation to current exposure limits (< 0.1 mg/m3), it is not uncommon in many industrial conditions. Several studies have estimated quartz burden in deceased coal miners, and quartz burdens of 200 mg/lung are considered to be low dose that are not associated with pneumoconiotic effects (29). As such, the dose rate used in this study was chosen to induce chronic pathologic effects in the rat, and will probably reflect a rapid development of silicosis in man.

Our results show a marked and time-dependent induction of the inflammatory response. The instillation of 2 mg of DQ12 resulted in a progressive increase in the total cell number, from the beginning of the observation time up to 90 d. A high percentage of PMN in the BALF (48%) were already observed 3 d after instillation and remained high until the end of our observation period (58%). The significant increase in total protein, as detected 28 d after instillation, indicates changes in vessel permeability and is supportive of a systemic influx of inflammatory cells into the lung. The patterns for ß-glucuronidase and MIP-2 in the BALF are indicative of an enhanced presence and/or activation state of inflammatory cells. In general, our BAL parameters indicate a marked persistence of the inflammatory process. Our observations indicate that increasing numbers of inflammatory cells, particularly macrophages, enter the lung, which may then become damaged, as indicated from the strong increase in LDH as observed at Day 90. Our data also show the induction of oxidative stress in the lung, as indicated from the increases in both TEAC and MPO. There is no linear correlation between the MPO activity (as a neutrophil specific marker) and the number of PMN, indicating that there is a difference between "presence" of the PMN, and the "activation" state for these cells. However, the correlation for TEAC versus protein concentration indicates that apparently most of the antioxidant capacity, as measured by the TEAC assay in the BALF, is "protein". This is supported by the fact that deproteinized lavage from the quartz treated rats did not show any differences from the controls. Surface modification appeared to be a very powerful tool in influencing the induction and persistence of the inflammatory response of quartz. The reducing effect of surface modification on the potential of quartz to generate ROS was demonstrated by EPR using the DMPO spin trap which enables measurement of •OH (30). We have shown previously, that surface coating of quartz inhibits its •OH-generating capacity, cytotoxicity, particle endocytosis and oxidative DNA damage in lung epithelial cells, as well as neutrophil burst activation in vitro (19). In the present study, we evaluated the stability of our quartz preparations during the experimental time in the storage tubes using EPR. The importance of the particle surface for ROS generation is reviewed by Fubini (5). Whereas PVNO is considered to bind to silanol groups on the silica particle, it is not yet fully understood by which mechanism the interaction between aluminum ions and the quartz surface reduces its activity (B. Fubini, personal communication). The fact that modification of the particle surface is an important tool to study biological particle effects in vivo was first demonstrated for AL coating by Begin and colleagues (31), who reduced the development of quartz induced fibrosis in sheep. Brown and coworkers (32) and Duffin and colleagues (22) inhibited the quartz-induced inflammatory reaction in rats in short time experiments. We are the first to show the inhibitory effects for both coatings on inflammation in a subchronic study, i.e., over a time frame of 90 d after instillation. Calculation of the amount of bound substances (11 µg PVNO/mg DQ12; 1.6 µg aluminum/mg DQ12) indicates that the inhibitory effects result from the surface modification of the quartz particle and is not due to effects of the substances per se. Subcutaneous application of PVNO has been used by other investigators to determine the effect of this compound on pulmonary effects of quartz (28), but was shown to be effective at only at much higher doses (20–140 mg/rat). In our current study, none of the evaluated parameters were found to be different between the control animals and the animals that were instilled with the coating substances.

PVNO surface modification showed a relatively stronger inhibitory effect on all BAL parameters compared with AL. Total cell numbers were reduced post instillation of both coated particles over the entire study period but small differences were measured in the percentage of PMN which, in the animals treated with the AL-coated quartzes, is significantly increased compared with PBS-treated animals. Increase in percentage of PMN in the DQ12+PVNO-treated animals started later, but was still significantly reduced at Day 90 compared with native silica–treated rats. Both coatings reduced the acute inflammatory and toxic effects by quartz, whereas PVNO also inhibited the persistency of the inflammation. Whether our current observations on toxicity, inflammation, and oxidative stress in this subchronic study are important for the pathogenicity of quartz will be evaluated using other chronic endpoints, including type II cell hyperplasia and fibrosis. In this regard, chemistry and stability of binding of the different coatings on the quartz surface is the subject of further investigations.

Because NF-{kappa}B is considered as a crucial transcription factor involved in inflammatory cellular responses (9), we investigated the activation of this pathway in our present study in relation to surface modification of the quartz particles. NF-{kappa}B activation was investigated by using two independent methods, which compared with a single assay is considered to provide much stronger support for the involvement of this pathway (33). First, levels of the cytosolic NF-{kappa}B inhibitor protein I{kappa}B{alpha} were measured in whole lung homogenates of particle-treated and control animals 3 and 90 d after instillation. Second, expression and translocation of the p65 subunit of NF-{kappa}B were determined in lung sections of these animals.

Both at 3 d and at 90 d after instillation, the I{kappa}B{alpha} levels tended to be reduced in the quartz-treated animals compared with the controls. At 90 d, this difference reached significance, indicative of a persistent activation of NF-{kappa}B. Although it is not known whether this effect is due to the quartz particles themselves or a result of particle-induced production of cytokines and/or ROS, our results are the first to show a persistent effect of quartz treatment on this inhibitor in vivo. Our current observations are also in line with in vitro studies showing persistent I{kappa}B{alpha} depletion in macrophages as well as in epithelial cells following treatment with quartz particles (26, 34). In contrast, PM10 (particulate matter)-induced NF-{kappa}B activation in vitro was found to occur in the absence of I{kappa}B{alpha} degradation (35). On the other hand, the fact that stronger effects were seen at the later time point in our current in vivo study suggests that inflammatory mediators may play a dominant role in the NF-{kappa}B activation pathway.

Immunohistochemical analysis revealed NF-{kappa}B activation in the form of p65 staining within the lungs of quartz-treated animals at all time points. Enhanced NF-{kappa}B activation and increased immunoreactivity of p65, the major transactivating member of the NF-{kappa}B family, has also been previously shown to occur in rat lungs during the development of asbestos-induced inflammation (36, 37). Interestingly, a high NF-{kappa}B signal was not only observed in alveolar macrophages, which is in concordance with the observed NF-{kappa}B activation in BAL cells as described by Duffin and coworkers (22) and Porter and colleagues (38), but also in bronchial as well as alveolar epithelial cells. Several in vitro studies have indeed shown that quartz particles can directly activate NF-{kappa}B in vitro (12, 39, 40). Importantly, in this regard, NF-{kappa}B activation has also been described to be involved in cell cycle regulation processes, and may as such play a role in the replacement of damaged epithelial cells (41). The active participation of epithelial cells after silica exposure is described in mice by Hubbard and coworkers (42). The focal occurrence of this activation at the early 3 d time point could also explain the lack of significance in the I{kappa}B{alpha} degradation as observed at the 3 d time point with quartz, i.e., due to a dilution effect of the whole lung homogenate. The significantly reduced I{kappa}B{alpha} levels at 90 d may relate to the severity and lung distribution of inflammation as observed at this time point. Earlier, Lentsch and colleagues (14) demonstrated in a nonparticle model that macrophages are crucial for pulmonary NF-{kappa}B activation and development of inflammatory processes. Driscoll and coworkers (11) demonstrated the role of epithelial cells in MIP-2 production and NF-{kappa}B activation by direct quartz exposure. Inhibition by antioxidant treatment suggests a role for ROS in particle-induced NF-{kappa}B activation. Synergism of ROS/RNS and TNF-{alpha} in the induction of NF-{kappa}B activation in rat lung epithelial cells has been shown by Janssen-Heininger and colleagues (10). Therefore, parallel to our in vivo analysis of NF-{kappa}B, in vitro experiments were performed to further unravel the possible mechanisms and cell type–specific involvement of activation of this pathway.

In the present study, we have demonstrated that the BALF from quartz-treated animals, as well as the supernatants from quartz-treated macrophages, activate NF-{kappa}B in rat lung epithelial cells in vitro. Our results are also in agreement with those of Jimenez and coworkers (15), who showed an increase in NF-{kappa}B binding in alveolar epithelial cells after treatment with supernatant from monocyte-derived macrophages activated with PM10. In these experiments involving PM10, the activation of NF-{kappa}B was discussed as a result of increased TNF-{alpha} production by the treated cells. The role of oxidant-induced TNF-{alpha} in the chemokine response of a murine lung epithelial cell line after cristobalite treatment has also been shown by Barrett and coworkers (43, 44) using a neutralizing antibody. In the present study, significant NF-{kappa}B activation in epithelial cells was only seen with TNF-{alpha} at a concentration of 1 ng/ml, which is at least 100-fold higher than that which might be present in the BALF from the quartz-treated animals (i.e., < 5 pg/ml). This suggests that apart from TNF-{alpha}, other constituents within the BALF were responsible for NF-{kappa}B activation in vitro. These observations were also supported from our in vitro observations on the lack of significant changes in NF-{kappa}B activation in epithelial cells after addition of anti–TNF-{alpha} antibody to our macrophage supernatants.

Whether this also holds true for our in vivo observations cannot be answered from our current data. Indeed, TNF-{alpha} has been causally linked to the development of quartz-induced fibrosis (45). From the contrasting effects of recombinant TNF-{alpha} versus TNF-{alpha} antibody on silica-induced fibrosis, a role for macrophage-derived TNF in inducing fibroblast proliferation and collagen production was suggested (45). However, the causal relation between TNF-{alpha} and particle-induced NF-{kappa}B activation and pulmonary inflammation is less clearly established. The observed lack in detectability of TNF-{alpha} in our study should take into account that our BAL samples were highly diluted (volume of 4 x 5 ml), and TNF-{alpha} concentrations in the local microenvironment of the lung might still have been high and therefore possibly play a role in the observed in vivo NF-{kappa}B activation. Importantly, however, and in line with our current in vitro and in vivo observations, Driscoll and colleagues (2) also failed to measure enhanced in vitro TNF-{alpha} production from lavage macrophages of rats that were instilled with quartz dosages which caused significant pulmonary inflammation, and which were similar to those used in our study. Furthermore, Ernst and coworkers (28) failed to show enhanced LPS-induced TNF-{alpha} production in vitro by BAL macrophages from animals that were instilled with 3 mg DQ12, compared with LPS-induced TNF-{alpha} production from BAL macrophages from saline-treated rats. Most importantly, it was recently shown in murine TNF-receptor knockout models by Ortiz and colleagues (46) that silica can cause NF-{kappa}B activation in vivo via TNF-independent pathways. Finally, TNF knockout mice versus wild-type mice were recently found to show no difference in extent of inflammation upon inhalation of diesel and carbon black particles (A. T. Saber, personal communication).

Various other constituents may have contributed to the possible TNF-independent NF{kappa}B activation in our present studies, including cytokines (e.g., IL-1), ROS (e.g., H2O2), or proteases, which have all been shown to activate NF-{kappa}B in vitro (47). An interesting candidate in our study is represented by MPO, which was clearly elevated in the BALF of the quartz-treated animals. Nys and coworkers (48) recently demonstrated a correlation between NF-{kappa}B activation and MPO activity present in BALF from patients with lung injury. Moreover, they showed that MPO can activate NF-{kappa}B in vitro. Finally, the enhanced oxidative stress as observed in the lungs from the rats using the TEAC assay also suggest excessive formation of ROS/RNS during inflammation, which may also impact on NF-{kappa}B activation. In this regard, hydrogen peroxide as well as 3-morpholinosyndronimine showed synergistic effects with TNF-{alpha} in activation of NF-{kappa}B in rat lung epithelial cells, and also suggests that signaling pathways elicited by ROS/RNS are different from TNF-{alpha}–induced signaling (49).

As already discussed, modification of the particle surface is considered to play a crucial role in the adverse effects of quartz, and has been suggested to relate to some of the observed variability in these effects in different industries (1, 5, 16). In the present study, we have clearly shown that modification of the particle surface with two independent coatings drastically impacts on pulmonary inflammation, oxidative stress, and toxicity. Our current data also indicate that the surface properties of quartz impact on acute as well as subchronic activation of the NF-{kappa}B pathway, which is considered to drive the observed inflammatory effects of quartz in our study. The role of the quartz-surface in NF-{kappa}B activation was shown in a number of independent ways, that is (i) in vivo analysis of lung sections and tissue, (ii) analysis of the effect of lavage fluid from treated animals, and (iii) analysis of conditioned media from particle treated macrophages on NF-{kappa}B activation. First, both the immunohistochemical appearance of NF-{kappa}B activation and the decrease in I{kappa}B{alpha} levels in whole lung homogenates, as observed in the quartz-treated animals, were found to be merely absent in the animals that were treated with the PVNO-modified quartz. These observations clearly corresponded to the marked reduction in subchronic inflammatory response observed in these animals. Interestingly, the lower efficiency of the AL surface modification to prevent quartz-induced inflammation as observed in our current study at 90 d, was accompanied by an increase in NF-{kappa}B activation and significant decrease in I{kappa}B{alpha} in the lungs of these animals. In line with our in vivo observations, surface modification of the quartz particles either with PVNO or with AL inhibited the NF-{kappa}B–activating potential of lavages of the quartz-treated animals, as well as the ability of macrophages to activate NF-{kappa}B after treatment with quartz. In summary, our complementary in vivo and in vitro observations with the different quartz preparations clearly show the importance of the particle surface in NF-{kappa}B–mediated acute and subchronic inflammatory effects of quartz.

In conclusion, we demonstrate that exposure to quartz leads to a persistent inflammatory response in the lung which is associated with NF-{kappa}B activation using in vivo and ex vivo approaches. We further demonstrate that the quartz surface plays a major role in both acute and chronic inflammation, toxicity, and oxidative stress in the rat lung. This postulated pathway may be crucial in the development of silica-induced diseases, such as fibrosis and cancer. Currently, epithelial proliferation and fibrosis are being evaluated in the same animal study by both classical and molecular pathology, and this may contribute to the understanding of the role of NF-{kappa}B in these processes. Our study also indicates that the reduced ability of surface modified quartz to activate NF-{kappa}B in vivo and in vitro may be due to various mechanisms including ROS generation and diminished toxicity to the epithelium. Finally, our data indicate that apart from TNF-{alpha}, other mediators are involved in the activation of an NF-{kappa}B–mediated response and associated induction of inflammation. Further studies are needed to elucidate the significance of TNF-independent mechanisms of particle-induced inflammation.


   Acknowledgments
 
The authors acknowledge the support of Silikose-Gesellschaft and Ministerium für Wirtschaft, Mittelstand, Technologie und Verkehr Nordrhein Westfalen (Germany). The authors thank Dr. Klaus Unfried from the Toxicology group of our Institute for his help with the instillation of the animals. They thank Mrs. Astrid Winzer, Christel Weishaupt, Kirstin Ledermann, and Veena Suri for their technical support in this extensive animal study. They are also grateful to Dr. Guido Haenen (Dept. of Pharmacology and Toxicology, Maastricht University, The Netherlands) for TEAC measurements in BALF. The authors thank Dr. Rodger Duffin for critically reviewing the manuscript.

Received in original form August 13, 2003

Received in final form April 7, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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