* Laboratory of Pneumology (Lung Toxicology), K. U. Leuven, Herestraat 49, B-3000 Leuven, Belgium; and
MRC Toxicology Unit, Lancaster Road, LE1 9HN, Leicester, United Kingdom
Received September 11, 2002; accepted November 25, 2002
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ABSTRACT |
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Key Words: polyvinyl chloride; PVC; additive; silica; particle; cytotoxicity; IL-8; IL-6; TNF-; A549; THP-1.
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INTRODUCTION |
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Dust exposure is often associated with airway inflammation. In chronic exposure the inflammation is generally recognized as having adverse effects and contributing fibrogenic mediators to lung fibrosis (Bassett and Bhalla, 2000). Both alveolar macrophages and epithelial cells act as a first-line defense against inhaled agents; they can recruit inflammatory cells to the airways via the release of chemo-attractants or cytokines. Interleukin-8 (IL-8), Interleukin-6 (IL-6), and tumor necrosis factor-
(TNF-
) mediate a variety of inflammatory effects in pulmonary inflammation. IL-8 and IL-6 are produced and released from both epithelial cells and alveolar macrophages, while the major source of TNF-
in the lung is alveolar macrophages (Lohmann-Matthes et al., 1994
; Martin et al., 1997
).
In a previous study, we tested 14 types of PVC particles and 16 relevant additives sampled from normal industrial processes, and found that some PVC particles produced by the emulsion process showed moderate in vitro cytotoxicity. The most toxic particles contained the most toxic additives (Xu et al., 2002). To elucidate the role of residual additives, two of these emulsion PVC particles were chosen for the present study, and they were compared with their "additive-free" counterparts and with the pure additives sodium lauryl sulfate and sodium alkylbenzenesulfonate. We investigated the cytotoxicity and the release of cytokines caused by PVC particles with or without additives in different relevant cells. Cytotoxicity was assessed in primary rat alveolar macrophages and type II pneumocytes and primary human alveolar macrophages (h-AM), as well as in cells from the human-derived A549 and THP-1 cell lines. Hemolytic potential was assessed in human erythrocytes. The effect on cytokine production was assessed in A549 cells, THP-1 cells, and h-AM.
We found that unwashed PVC particles exhibited the potential to damage pulmonary cells and to induce the release of inflammatory mediators in vitro. These particles might also have hazardous potential to the lung under excessive occupational exposure. The present study proposes that the hazardous potential of these PVC particles is most probably related to their residual additives.
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MATERIALS AND METHODS |
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Min-U-Sil (crystalline SiO2), kindly provided by Prof. B. Fubini (Facoltà di Farmacia, Università di Torino, Italy), was used as a positive particle control. The median size of particles was around 2 µm, as measured by means of a Coulter LS particle size analyzer at Vlaamse Instelling voor Technologisch Onderzoek (VITO), Belgium.
Samples for electron microscopy were dispersed in ultrafiltered ethanol, using a vortex mixer. Droplets of the resulting suspension were applied to aluminum specimen holders, dried-down, sputter-coated with gold, and examined in an ISI DS130 scanning electron microscope. The remaining suspension was mixed (1 + 1) with ultrafiltered, ultrapure water and mixed again. Droplets of this suspension were applied to nickel grids, precoated with a film of formvar/carbon, and examined in a Zeiss 902A transmission electron microscope.
Reagents and solutions.
Waymouths 752/1 medium, RPMI 1640 medium, penicillin-streptomycin solution (10,000 units, 10,000 µg/ml), fungizone (amphotericin 250 µg/ml), L-glutamine (200 mM), HEPES, and fetal bovine serum (FBS) were purchased from GibcoBRL company (N.V. Life Technologies, Merelbeke, Belgium). DMSO (dimethyl sulfoxide), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Percoll, lipopolysaccharide (LPS), polymyxin B sulfate, polyoxyethylenesorbitan monolaurate (Tween 20), trypsin type I (EC3.4.214. cat. No. T-8003), and phorbol myristate acetate (PMA) were purchased from Sigma Belgium (Bornem, Belgium). Deoxyribonuclease (DNAse) I was purchased from Boehringer Mannheim GmbH (Brussels, Belgium). All cell culture plastics were purchased from Corning. Sodium diethylbarbiturate (veronal), diethylbarbituric acid, and all other chemicals were purchased from VEL (Leuven, Belgium). Stock veronal buffer, veronal-buffered saline (VBS), and phosphate-buffered saline with calcium and magnesium (PBS+) were prepared as described in the previous study (Xu et al., 2002).
A549 cell line.
The A549 cell line is a human type II pneumocyte-derived cell line (Lieber et al., 1976), purchased from ATCC (American Type Culture Collection). Cells were incubated (37°C, 5% CO2) in Waymouths medium containing 10% FBS, 1% glutamine, 1% penicillin-streptomycin, and 0.5% fungizone in flasks. Cells were transferred to 96-well plates (100,000 cells/200 µl/well) 24 h before exposure to the test substance. Cell viability was greater than 95% when determined by trypan blue exclusion.
THP-1 cell line.
THP-1 is a human monocytic and nonadherent cell line. Under the influence of phorbol esters, THP-1 cells differentiate into adherent macrophage-like cells. This cell model can be employed to study the secretory function of the macrophage (Auwerx, 1991).
THP-1 cells were obtained from ECACC (Salisbury, UK; catalog nos. 8601284 and 88081201). The nonadherent monocytes were cultivated in RPMI 1640 with 25 mM HEPES, supplemented with 10% FBS, 1% penicillin-streptomycin, 1% glutamine, and 0.5% fungizone. The cells were transferred to 96 wells and incubated with 0.1 µg/ml PMA for 48 h before exposure to the test substance, to allow adherence and differentiation into mature macrophages. From here on, THP-1 cells refer to the matured and differentiated cells (i.e., macrophage-like).
Primary rat alveolar macrophages (r-AM) and rat type II pneumocytes (r-TII).
Male Wistar rats with a mean weight of 247 g (range: 231261 g) were obtained from the Janvier Co., the Netherlands. The rats were maintained in a conventional animal house, with 12-h light/dark cycles in cages with a wired bottom, and with free access to water and laboratory food. Cells were isolated as described previously (Richards et al., 1987). On average 58x106 macrophages (95% purity) and 15x106 type II cells (90% purity) were obtained per rat. Cells were transferred to 96-well plates (100,000 cells/200 µl/well) and incubated in the same medium as for A549 cell culture. Twenty h after plating, test samples were added to AM cultures. TII were supplied with fresh medium 24 h after plating, and 20 h later (i.e., 44 h after plating) test samples were added when the cell culture reached confluence.
Primary human alveolar macrophages (h-AM).
Human surgical specimens were obtained from the nontumoral part of the lung of three patients undergoing lobectomy for lung cancer. The ages of the patients were 59, 55, and 71 years and the Ethical Committee of the Faculty of Medicine approved the use of such tissue. Alveolar macrophages were isolated based on the methods described previously (Hoet et al., 1999; Xu et al., 2002
). The tissue was first sliced and then washed with NaCl (0.9%). The washing fluid contained mainly macrophages and erythrocytes. In order to eliminate the erythrocytes, the washing fluid was suspended in an isotonic shock solution containing 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA, pH 7.4 (Roos and Loos, 1970
), for 10 min at 4°C. The remaining cells were washed three times in PBS+. This yielded 510 x106 alveolar macrophages (more than 92% pure) per gram of tissue with a viability of more than 95%. Cells were transferred to 96-well plates (100,000 cells at 200 µ/well) and incubated in the same medium as for A549 cell cultures. Four h after plating, test samples were added to the cultures.
Human red blood cells (h-RBC).
The cells were obtained, as in our previous study (Xu et al., 2002), from volunteers by venipuncture, using Vacutainers® (Becton Dickson Co.) containing heparin. The serum and white cells were removed and the erythrocytes were washed three times with veronal-buffered saline (VBS). H-RBC were suspended at approximately a 2% concentration in VBS. In order to avoid donor differences, the initial h-RBC suspensions were adjusted to equal hemoglobin concentrations (Nolan et al., 1981
).
Exposure of adherent cells (A549, r-AM, r-TII, h-AM, THP-1) to particles and additives.
The particles/additives were freshly (i.e., 12 h before addition to the cells) suspended/dissolved in vehicle (Waymouth medium or RPMI 1640 medium + 5% ethanol), then vortexed for 1 min and placed in an ultrasonication bath (Branson 1200, Branson Ultrasonics Corp., working frequency: 47 kHz, HF-output power: 30 W, 15 min). The suspensions/solutions were pipetted into the cell culture (20 µl of sample + 180 µl culture medium per well); the wells containing control cells received 20 µl of vehicle and 180 µl culture medium. Final working concentrations were 2.50.004 mg/ml (1 mg/ml = 0.2 mg/105 cells = 6 µg/mm2) for particles and 0.040.0025 mg/ml for additives, with 4- or 5-fold dilution steps. During exposure of the cells to test compounds, the culture medium was serum-free (Waymouths medium or RPMI 1640 medium + 1% glutamine + 1% penicillin-streptomycin + 0.5% fungizone). Cytotoxicity was determined after 2024 h or 48 h of incubation. Aliquots of A549 cell culture supernatant (after 4-, 16-, 24-, and 48-h exposure), THP-1 cell culture supernatant (after 48-h exposure), and h-AM cell culture supernatant (after 24-h exposure) were frozen at -80°C for subsequent determination of IL-8, IL-6, or TNF- concentrations.
Exposure of h-RBC to particles and measurement of hemolysis.
The particles were suspended in EV (VBS containing 10% ethanol) on the day of the experiment. A volume of 1 ml h-RBC suspension was added to the glass tubes containing 3 ml sample suspension. The mixtures were placed in a water bath (37°C) for a period of 2 h. The tubes were inverted every 30 min by hand to resuspend the settled cells and particles. At the end of 2 h incubation, all tubes were centrifuged (1600 g) for 20 min and the absorbance of the supernatant was determined.
Background controls (1 ml h-RBCS + 3 ml EV = 0% lysis) and total lysis controls (1 ml h-RBCS + 3 ml distilled water = 100% lysis) were treated identically in each experiment. Working concentrations were 0.25, 0.5, 1, 2, and 4 mg/ml.
The absorbance of liberated hemoglobin, or hemolysis, was recorded at 540 nm, using a Beckman Model DU-65 spectrophotometer. Viability was expressed as 100% minus the percentage hemolysis versus vehicle control.
Measurement of cellular toxicity in adherent cells.
As an index of cell toxicity, the decline in MTT reduction was measured (Mosmann, 1983). MTT solution (0.5 mg/ml) was added to each culture well (200 µl/well) and incubated for 1 h (37°C, 5% CO2). Then the formation of the purple formazan was assessed spectrophotometrically (Benchmark Microplate Reader, Bio-Rad) at 550 nm, with reference wavelength 650 nm. Viability of cells was expressed as a percentage versus vehicle control.
Measurement of cytokine (IL-8, IL-6, and TNF-a) production.
Cytokine proteins in the cell culture supernatants released from treated A549 cells, THP-1 cells, or h-AM were measured by ELISA using kits from BIOSOURCE (HU IL-8 cytoset, HU IL-6 cytoset, and HU TNF- cytoset). Each assay was performed according to manufacturers directions and read on a Bio-Rad microplate reader at 450 nm (with reference filter at 655 nm). Standards and samples were pipetted into 96-well microplates (NUNCTM, Nunc-ImmunoTM, Module, MaxiSorpTM surface) precoated with coating antibody, followed immediately by addition of biotinylated antibody. After a room temperature incubation step, unbound antigen was washed off and streptavidin-horseradish-peroxidase conjugate was added. Following another incubation and wash step, substrate was added and color development was in proportion to the amount of cytokine added to the wells. The lower limits of detection for IL-8, IL-6, and TNF-
were 8.2, 10.2, and 10.2 pg/ml, respectively.
Endotoxin measurements.
Endotoxin measurements of the tested particles were performed using the Limulus Amebocyte Lysate (LAL) assay (E-TOXATE Kit, Sigma).
Data presentation and analysis.
At least 3 separate experiments were performed with each substance, concentration, and cell system. The results were analyzed with Prism 3.0 Graphpad software (San Diego, CA). Statistical differences were assessed by two-way analysis of variance (ANOVA). Two-tailed tests of significance were employed and significance was assumed at p < 0.05.
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RESULTS |
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Reduced cytotoxicity and hemolytic activity with additive-free particles.
Confirming our previous findings (Xu et al., 2002), significant cytotoxicity and hemolysis were observed for PVC particles E3 and E8, and their relevant additives (A3 and A8) in the different cells used. As shown in Figure 2
, the cytotoxicity and hemolytic activity of E3 and E8 particles were abolished or markedly decreased after removal of their additives.
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Cytokine release from h-AM.
The evaluation of cytokine release from h-AM was complicated by variability between and within patients, although the cell viability experiments were consistent across the three batches of macrophages (Fig. 5). Within patients, the variability in cytokine release was higher and led to SD of up to 45% in IL-8 release in control wells (n = 8). Macrophages from patient 1 had no IL-8 response to Min-U-Sil and PVC particles, cells from patient 2 appeared to "overreact" to all concentrations of Min-U-Sil and PVC particles, while only the cells from patient 3 showed a "decent" dose-response reaction to Min-U-Sil. The variability in response to the particles precluded any meaningful statistical analysis and interpretation, and this is why we chose to work with a macrophage cell line.
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Two concentrations of LPS (0.25 and 5 EU/ml) were applied in the experiments to examine the effect of LPS on cytokine release from the cells. At these concentrations, LPS did not induce cytotoxicity in A549 or THP-1 cells. No significant increases of IL-6 or TNF- release from A549 cells were found after 448 h of exposure to LPS. However, the release of IL-8 was significantly increased after 24 or 48 h of exposure to LPS at 5 EU/ml, but not at 0.25 EU/ml, which is higher than the highest endotoxin level found in our particle suspensions (Table 1
). We also tested the effect of suspending the particles with polymyxin B (10 µg/ml) to neutralize the effect of LPS (Dong et al., 1996
; Morrison and Jacobs, 1976
) before addition to the cell cultures, but the cytokine release remained similar (data not shown). Neither measurable IL-6 release nor significant decrease of cell viability were found in THP-1 cells after a 48-h exposure to LPS. However, the release of TNF-
and IL-8 were significantly increased after 48 h of exposure to LPS at 5 EU/ml, but again not at 0.25 EU/ml (Table 1
). Thus, we concluded that any increased cytokine release after exposure to particles was not due to their possible endotoxin contamination.
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DISCUSSION |
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Cytokine measurement and cell cultures.
IL-8 is an essential factor for neutrophil infiltration in most inflammatory reactions (Sekido et al., 1993). However, according to a recent review "no clear-cut IL-8 homolog in rat or mouse has been identified so far, although macrophage inflammatory protein-2 (MIP-2) and KC may be functional homologs of IL-8 in mice" (Iizasa and Matsushima, 2000
, 1065). In the rat there are at least three members of the CXC chemokine family that are potent chemotactic factors for neutrophils: cytokine-induced neutrophil chemoattractant-1 (CINC-1), CINC-2 and CINC-3/ MIP-2 (Shibata et al., 2000
). Not all above-mentioned cytokine assays are available for rat. Therefore, we preferred to assess the release of cytokines from human cells in the present study. We have attempted to assess this in primary human alveolar macrophages obtained from surgical specimens, as in our previous study (Xu et al., 2002
). Figure 5
shows that although the cell viability experiments were consistent across the three batches of macrophages that we had isolated from three patients, there were large variations in cytokine release within and between individual subjects. Such variability is not unusual when working with patient-derived tissues. However, because this variability complicated the interpretation of our results, we chose to use more stable cell lines in the present study.
Endotoxin level and cytokine release.
Endotoxin is known to have an effect on induction of cytokine production (Thorne et al., 1999), but it was not responsible for the induction of the cytokine release by cells exposed to PVC particles or silica in our study, since we demonstrated that LPS at 0.25 EU/ml, which is higher than the endotoxin level found in our particle suspensions, could not induce cytokine release from A549 cells or THP-1 cells. Nevertheless, the elevated release of cytokines by both types of cells after exposure to a higher LPS concentration (5 EU/ml) verified that LPS could effect an inflammatory response.
Differential cytokine release in response to particles and additives: Comparison of A549 cells and THP-1 cells.
Cytokine release, upon exposure to particles, exhibited a nonlinear dose-response relationship. At a concentration of 0.156 mg/ml, Min-U-Sil led to the highest enhancement of cytokine release in both A549 cells and THP-1 cells (Figs. 4 and 6). However, less than half of the cells survived at higher concentrations, as assessed by the MTT assay. Consequently the apparent decrease of cytokine production at higher concentrations is most likely an effect of fewer cells available to produce it. This may also explain the decreased cytokine level in THP-1 cell cultures after exposure to cytotoxic concentrations of particles and additives (Fig. 6
).
The additives are highly toxic, but the original PVC particles only contain less than 1% of these additives. The responses of cells after treatment with PVC particles or the additives alone were qualitatively similar, but the magnitude of difference between effective concentrations of PVC particles and their additives was approximately 100-fold lower. Limited experiments (data not shown) were performed using the supernatant obtained after centrifugation of suspended PVC particles. Supernatant alone induced slight toxicity but the major toxic effect remained with resuspended particles, indicating that the additives were not readily released into the medium. In fact, even after four successive methanol washings, 58% of the additives still remained associated with the particles.
The cytokine release from A549 cells was induced by PVC particles and Min-U-Sil, but only Min-U-Sil induced cytokine release from THP-1 cells (Figs. 4 and 6). The magnitude of the increases in IL-6 and IL-8 release after particle exposure in A549 cells is significantly greater. Also after exposure to highly toxic Min-U-Sil, A549 cells still produced more cytokines, but THP-1 cells did not. We speculate that A549 cells have a stronger ability to produce cytokine in response to particles than THP-1 cells, and alveolar epithelial cells respond in a cell-specific way to this type of trigger. It is possible that the potential of THP-1 cells to produce additional cytokines was exhausted by their prior incubation with phorbol esters, thus resulting in a less marked response to the subsequent particle exposure. The IL-8 and TNF-
levels in control THP-1 cell supernatants were more than 20 times higher than those of control A549 cells.
Comparison of PVC particles with Min-U-Sil.
The induction of cytokine release was rapid by Min-U-Sil, with cytokine protein elevated 4 h after exposure, and it was also more potent, except for IL-6 (Figs. 4 and 6). This is compatible with the notion that crystalline silica is one of the most fibrogenic dusts (Fubini, 1997
). However, we were surprised to find that the magnitude of the increase in IL-6 release by A549 cells exposed to PVC E3 or E8 particles was significantly greater than after exposure to Min-U-Sil. IL-6 acts on proliferating B cells to promote differentiation into antibody forming cells (plasma cells) and it stimulates antibody secretion. It has been described as a "hepatocyte stimulating factor" and strongly stimulates hepatocytes to make acute-phase proteins in response to inflammation (Zhang et al., 1995
). It is also an effective inducer of tissue factor upregulation and may trigger endothelial cells to change their antithrombotic properties into a procoagulant, clot-promoting state (Grignani and Maiolo, 2000
). IL-6 stimulates fibrinogen expression both in vivo and in vitro (Castell et al., 1990
; Rokita et al., 1993
). The apparently specific effect of PVC particles on IL-6 production and its relevance for pulmonary disease remain to be explored, particularly with regard to granulomatous lung disease.
Relevance of the study.
In humans, PVC pneumoconiosis has some features of granulomatous lung disease (Antti-Poika et al., 1986; Arnaud et al., 1978
; Studnicka et al., 1995
; Szende et al., 1970
). Rats developed focal pulmonary fibrosis after a 15-week inhalation of PVC dust at a "nuisance" level, i.e., 10 mg/m3 (Richards et al., 1981
). Inflammation is likely to be part of the mechanisms, although this was not investigated in the study. The initial steps of the airway inflammation include stimulation of resident cells, release of chemotactic agents, and recruitment of inflammatory cells, which ultimately results in the development of chronic lung disease (Bassett and Bhalla, 2000
). In the present study, unwashed PVC particles (E3 and E8) exhibited the potential to damage pulmonary cells and to induce the release of inflammatory mediators, such as IL-8 and IL-6. IL-8 enhances inflammation, by enabling immune cells to migrate into tissue (Huber et al., 1991
). IL-6 plays a critical role in the progress of lung inflammation/injury (Yu et al., 2002
). Therefore, excessive occupational exposure to these PVC particles might cause cellular damage and chronic inflammation. According to our in vitro study, reducing the amount of residual toxic additive might prevent these processes. Additional studies in vivo will follow to investigate whether these PVC particles can induce lung inflammation and whether the in vivo response is correlated with the in vitro results.
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ACKNOWLEDGMENTS |
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NOTES |
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