Introduction

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Recent epidemiologic studies have shown an association between cardiopulmonary morbidity and mortality and levels of ambient particulate matter with a diameter of less than 10 µm (PM₁₀) (1, 2). Residents of communities exposed to high compared with low levels of air pollution have faster rates of decline in lung function, more chronic respiratory and cardiovascular disease, and hospital admissions for pneumonia, chronic obstructive pulmonary disease (COPD), myocardial infarction, and heart failure after adjusting for several individual risk factors, including cigarette smoking (3).

Previous studies in our laboratory showed that small inert carbon particles instilled into the lungs of rabbits induced a systemic inflammatory response that includes stimulation of the bone marrow (4). This observation was supported by a report from Tan and colleagues demonstrating a leukocytosis in young military recruits during an episode of acute air pollution in Southeast Asia (5). This leukocytosis was associated with bone-marrow stimulation and greater release of granulocytes from the bone marrow into the circulation (5). Stimuli such as acute pneumonia (6), endotoxemia (7), and cigarette smoke exposure (8) increase the number of younger immature granulocytes in the circulation. These immature granulocytes preferentially sequester in the lung microvessels, and activation of these sequestered cells by local or circulating inflammatory mediators is thought to damage the endothelium (9).

Alveolar macrophages (AM) and lung epithelial cells are directly exposed when small atmospheric particles are inhaled. Several studies have shown that PM₁₀ stimulates the production of reactive oxygen species and inflammatory mediators from bronchial epithelial cells (10). Airway epithelial cells can also synthesize and express a variety of proinflammatory cytokines, such as interleukin (IL)-1, IL-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-, and granulocyte macrophage colony-stimulating factor (GM-CSF), that can influence the airway inflammatory response and contribute to the airway lesions in asthma and COPD (13).

This study was designed to test the hypothesis that the exposure of bronchial epithelial cells to PM₁₀ results in the expression of key genes involved in producing cytokines that have the ability to elicit a systemic inflammatory response that includes stimulation of the bone marrow. We used primary cultures of human bronchial epithelial cells (HBECs) freshly isolated from lobectomy or pneumonectomy specimens. Both a ribonuclease (RNase) protection assay (RPA) that can simultaneously quantify several messenger RNA (mRNA) species in a single sample of total RNA and enzyme-linked immunosorbent assays (ELISAs) were used to assess cytokine production by HBECs. We studied the effects of both these particles and the soluble fraction of the particles on cytokine production by bronchial epithelial cells.

	Materials and Methods

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Particles (PM₁₀)

The PM₁₀ particles (EHC-93) were collected by Environmental Health Directorate, Health Canada (Ottawa, ON, Canada). A detailed analysis of the EHC-93 has been presented elsewhere (14). Briefly, the particles were recovered from videlon bag filters, with a nominal cutoff of 0.3 µm, from a single-pass filtration system of the Environmental Health Center (EHC) in Ottawa, ON, Canada, with no recirculation of indoor air. The material was sieved through a 36-µm mesh nytex monofilament filter. This dust sample has a particle diameter of 0.8 ± 0.4 µm (median ± standard deviation) with 99% of particles < 3.0 µm. It contains sulfates and various metals; by elemental analysis, the most abundant are aluminum, calcium, copper, iron, lead, magnesium, sodium, tin, titanium, and zinc. Particles were suspended in hydrocortisone-free bronchial epithelial cell growth medium (BEGM) (Clonetics, San Diego, CA) at a concentration of 1 mg/ml and sonicated three times for 1 min each time at maximal power on a Vibra Cell VC-50 sonicator (Sonics & Materials, Inc., Danbury, CT) before the cells were added. In a separate set of experiments, the PM₁₀ suspension of the 100-µg/ml preparation was filtered through a Syringe filter (pore size: 0.22 µm) (Corning, Cambridge, MA) to obtain a clear solution. The endotoxin content of the PM₁₀ suspension after sonication was measured using a commercially available kit (Limulus Amebocyte Lysate Test, QCL-1000; BioWhittaker, Inc., Walkersville, MD) according to the manufacturer's instructions. Carbon particles (colloidal carbon [CC]; Fount India Drawing Ink, Pelikan, Germany) were suspended in hydrocortisone-free BEGM. The average size of these carbon particles was less than 1 µm as measured by flow cytometry, and the concentration of endotoxin in the CC was less than 0.015 EU/ml by Limulus test (E-TOXATE; Sigma Chemical Co., St. Louis, MO).

Cell Culture

Bronchial tissue was obtained from four patients who underwent lobectomy or pneumonectomy for lung cancer at St. Paul's Hospital (Vancouver, BC, Canada). All were current smokers with a mean age of 64.3 yr (range 48 to 70 yr). Lung segments away from and free of the tumor were used. Primary HBECs were isolated from these bronchial tissues according to a previously described procedure (15). In brief, pieces of excised human bronchial tissue approximately 1 cm long were incubated at 4°C for 24 h with 0.1% protease (Type 14; Sigma) solution prepared in Eagle's minimum essential medium (GIBCO BRL, Gaithersburg, MD) containing Fungizone (1 µg/ml; GIBCO BRL). The epithelial cells were harvested, washed with BEGM with added antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin; Sigma) and Fungizone, and cultured in a 25-cm² cell culture flask (Corning) until 80 to 90% confluent. Then the cells were trypsinized and placed in 100-mm cell culture dishes (Falcon; Becton Dickinson Ltd., Mississauga, ON, Canada) and cultured in BEGM. The purity and identity of the cells were checked morphologically using light microscopy.

Exposure of Cells to PM₁₀

HBECs were passaged at 80 to 90% confluence and all experiments were performed at either the third or fourth passage. When the cells were 90 to 100% confluent in 100-mm cell culture dishes (approximately 2.5 to 3.0 × 10⁶ cells/dish), hydrocortisone was removed from the cell culture medium 24 h before treatment and during the time of the study. Fresh stock suspensions of PM₁₀ (EHC-93) were prepared in BEGM without hydrocortisone before each experiment and added to 10 ml of culture medium to give final concentrations of 10, 100, and 500 µg/ml. To examine the biologic activity of the soluble fraction, PM₁₀ solution (100 µg/ml) filtered as described earlier was added to the cells. To examine the effect of endotoxin, lipopolysaccharide (LPS) (Escherichia coli 0111:B4; Sigma) was dissolved in sterile distilled water at 10 mg/ml, stored at 70°C and diluted with BEGM without hydrocortisone before use, and added to culture medium for a final concentration of 1 µg/ml. To examine the effects of inert particles, CC was suspended in hydrocortisone-free BEGM and added to 10 ml of culture medium to give a final concentration of 1%. To assess whether the HBECs phagocytose PM₁₀ particles, cells were grown to confluence and exposed to 100 µg/ml of PM₁₀ suspension for 24 h. Cell cultures were harvested by brief trypsinization and centrifugation. The pellet, fixed in 2.5% glutaraldehyde for 1 h, was mixed with 1% agarose and embedded in JB-4, and sections of 2 to 3 µm were cut. The preparations were stained with hematoxylin and eosin (H&E) and particles identified using a light microscope at ×800 magnification. Cell viability was determined after 24 h PM₁₀ exposure in all experiments using the trypan blue exclusion method.

RPA

Total RNA was isolated from harvested cells using a single-step phenol/chloroform extraction procedure (Trizol; Life Technologies, Inc., Grand Island, NY). Cytokine mRNA levels were determined using an RPA RiboQuant multiprobe system (PharMingen, San Diego, CA) following the instructions of the supplier. Two template sets were used. One set (hCK-4) was for human IL-3, IL-7, GM-CSF, macrophage (M)-CSF, granulocyte (G)-CSF, IL-6, leukemia inhibitory factor (LIF), stem cell factor (SCF), and oncostatin M (OSM). The second was a customized template set for human regulated on activation, normal T cells expressed and secreted (RANTES), IL-12p40, TNF-, IL-1, macrophage inflammatory protein (MIP)-1, MCP-1, IL-8, IL-18, and interferon (IFN)-. These template sets also included ribosomal protein (L32) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. In brief, 10 µg of total cellular RNA was hybridized overnight to the [³²P]uridine triphosphate-labeled riboprobes that had been synthesized from the supplied template sets. Single-stranded RNA and free probe remaining after hybridization were digested by RNase A and T1 mix. The protected RNA was then phenolized, precipitated, and analyzed on a 5% denaturing polyacrylamide gel. After electrophoresis, the gel was dried and subjected to autoradiography and the quantity of protected RNA was determined using densitometry and NIH image 1.62 (National Institutes of Health, Bethesda, MD) software. Results were normalized to the expression of the internal control, GAPDH.

ELISA for LIF, IL-8, IL-1, and GM-CSF

Cell culture supernatants were collected 24 h after addition of 100 to 500 µg/ml of PM₁₀ suspension, soluble fraction of 100 µg/ml PM₁₀, or 1 µg/ml of LPS, then centrifuged and stored at 70°C until measured by ELISA. The concentrations of human IL-1 and GM-CSF were measured by the Cytokine Core Laboratory (Baltimore, MD) using a biotin-strepavidin-peroxidase detection system. All measurements were done in triplicate and values reported are the means of the four measurements. For measurement of human LIF and IL-8, ELISA plates (Corning) were coated with 100 µL/well of the capture antibody (Ab) (monoclonal mouse antihuman LIF Ab at 2.5 µg/ml or monoclonal mouse antihuman IL-8 Ab at 4 µg/ml; R&D Systems, Minneapolis, MN) in phosphate-buffered saline overnight. After treatment with a blocking buffer, 100 µl of samples and standard (recombinant human LIF or IL-8) were incubated in combination with 100 µL/well of the detection Ab (biotinylated goat antimouse LIF Ab at 100 ng/ml or biotinylated goat antimouse IL-8 Ab at 20 ng/ml; R&D Systems). After incubation with streptavidin-horseradish peroxidase (ZYMED, San Francisco, CA) and substrate solution (a mixture of tetramethylbenzidine dyhidrochloride and H₂O₂), the reaction was stopped by adding 50 µl of stop solution (1 M H₂SO₄). Plates were read on a Spectra and Rainbow Reader (SLT Lab Instruments, Grödig, Austria) at 450 nm and the cytokine concentration in each sample was calculated from the standard curve. All measurements were performed in triplicate and values reported are means of the four measurements. The detection ranges of LIF, IL-1, GM-CSF, and IL-8 were 23.4 to 1,500, 3.125 to 200, 12.5 to 800, and 31.2 to 2,000 pg/ml, respectively.

Statistical Analysis

Data are expressed as mean values ± standard error. The minimum number of replicates for all measurements was at least three. Differences between multiple groups or over time were compared by one-way analysis of variance. The post hoc test used was Fisher's PLSD test. Differences between two groups of the CC experiments were compared by Mann-Whitney U test. Significance was assumed at P < 0.05.

	Results

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Cell Culture

HBECs incubated with 100 µg/ml of PM₁₀ suspension for 24 h phagocytosed the PM₁₀ particles as shown in Figure 1. A total of 18.6% of cells internalized particles, and of those, most contained more than one particle. The PM₁₀ were not toxic to the HBECs and > 90% of cells were viable after 24 h exposure to up to 500 µg/ml PM₁₀. The EHC-93 is known to contain small amounts of LPS (12), and the endotoxin content of the PM₁₀ suspension of 100 µg/ml was 6.4 ± 1.8 EU/ml (n = 4).

View larger version (129K): [in this window] [in a new window]   Figure 1.   Photomicrograph of a primary culture of HBEC incubation with 100 µg/ml of PM10 (EHC-93) for 24 h, showing particles internalized by the epithelial cells (arrow). Cells embedded in plastic were cut as sections 2 to 3 µm thick and stained with H&E. The bar represents 10 µm.

Effect of PM₁₀ on Expression of Cytokine mRNA

Figures 2A (the hCK-4 template set) and 2B (the customized template set) show the cytokine mRNA expression using RPA by HBECs at 2, 8, or 24 h incubated with medium alone (control); 10, 100, and 500 µg/ml of PM₁₀ suspension; soluble fraction of 100 µg/ml PM₁₀; and 1 µg/ml of LPS. Figure 2A shows that LIF mRNA expression was increased after 2 h incubation with PM₁₀ in a dose-dependent manner and that the levels after incubation with 100 and 500 µg/ml of PM₁₀ were significantly higher compared with control values (P < 0.01). LIF mRNA expression decreased after 8 and 24 h of exposure to PM₁₀. LPS was a weak inducer of LIF mRNA expression in HBECs. Figure 2A also shows that mRNA expression of GM-CSF in HBECs increased after 8 h of exposure to PM₁₀, with a maximum increase at 24 h (P < 0.01), in a dose-dependent manner. LPS caused an early (2 h) and a late (24 h) induction of GM-CSF mRNA expression (P < 0.05).

View larger version (100K): [in this window] [in a new window]   View larger version (97K): [in this window] [in a new window]   Figure 2.   mRNA expression of several cytokines after incubation for 2, 8, and 24 h in medium alone (control; lane 1), 10 µg/ml PM10 (lane 2), 100 µg/ml PM10 (lane 3), 500 µg/ml PM10 (lane 4), the soluble fraction of 100 µg/ml PM10 (lane 5), and 1µg/ml LPS (lane 6). GAPDH was used to correct for lane loading conditions. Expression was quantified using densitometry. Results shown in the bar graph are the percentage change from control (medium alone) values at the time point of maximum cytokine mRNA expression. Values are means ± standard error of the mean (SEM) of four experiments. *P < 0.01, **P < 0.05 compared with control. (A) Cytokine mRNA expression in HBECs using the hCK-4 template set. LIF mRNA expression increased within 2 h of exposure to PM10 in a dose-dependent manner. The maximum response was seen at 2 h (*P < 0.01). The soluble fraction of PM10 induced significantly less mRNA expression than did the whole particles at 2 h (P < 0.05), and LPS caused a weaker LIF mRNA response than either 100 or 500 µg/ml PM10 (P < 0.01). GM-CSF mRNA from HBECs was constitutively expressed and increased in a dose-dependent manner after 8 and 24 h of exposure to PM10, with a maximum response at 24 h (*P < 0.01). LPS caused an early (2 h) and a late (24 h) increase of GM-CSF mRNA expression (**P < 0.05). mRNAs for IL-3, IL-7, G-CSF, SCF, and OSM were not detected. Cytokine mRNA expression in HBECs using a customized template set. IL-1 mRNA expression in HBECs was constitutively expressed and showed a dose-dependent increase to PM10 exposure at 8 and 24 h with a maximum response at 24 h (*P < 0.01). The soluble fraction of PM10 induced significantly less IL-1 mRNA expression than did the whole particles at 24 h (P < 0.05), and LPS caused a weaker response than did 500 µg/ml PM10 (P < 0.05). IL-8 mRNA expression in HBECs increased within 2 h of exposure to PM10, with a dose-dependent increase at 8 and 24 h. The maximum response was seen at 8 h (*P < 0.01). The soluble fraction of PM10 induced significantly less IL-8 mRNA expression than did the whole particles at 8 h (P < 0.05), and LPS caused a weaker response than did 500 µg/ml PM10 (P < 0.01). mRNAs for RANTES, IL-12, TNF-, MIP-1, and IFN- were not detected.

Figure 2B shows that mRNA expression of IL-1 in HBECs increased after 2 h of exposure to PM₁₀ with a dose-dependent increase at 8 and 24 h and a maximum response at 24 h (P < 0.01). LPS caused a weaker mRNA response than did 100 or 500 µg/ml PM₁₀. Figure 2B also shows that IL-8 mRNA expression in HBECs increased after 2 h of exposure to PM₁₀ with a dose-dependent increase at 8 and 24 h and a maximum response at 8 h (P < 0.01). LPS caused a weaker mRNA response than did 100 or 500 µg/ml PM₁₀.

There was a small, nonsignificant increase in IL-6 mRNA expression of HBECs incubated with 100 and 500 µg/ml PM₁₀ (Figure 2A). M-CSF (Figure 2A), MCP-1 (Figure 2B), and IL-18 (Figure 2B) mRNA were constitutively expressed with no difference between controls, PM₁₀, or LPS. Neither PM₁₀ nor LPS induced HBEC mRNA expression of IL-3, IL-7, G-CFS, SCF, OSM, RANTES, IL-12, TNF-, MIP-1, and IFN- (Figure 2).

The HBECs exposed to the soluble fraction of 100 µg/ml PM₁₀ produced levels of mRNA for LIF, GM-CSF, IL-1, and IL-8 similar to the control (Figure 2). LIF, IL-1, and IL-8 mRNA expression after stimulation with the soluble fraction of 100 µg/ml was also significantly lower than that achieved after stimulation with 100 µg/ml of PM₁₀ suspension at 2, 24, and 8 h, respectively (P < 0.05; Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Exposure to the soluble fraction of 100 µg/ml PM10 (open bars) induced significantly less LIF, IL-1, and IL-8 mRNA expression than did 100 µg/ml of the whole particles (filled bars) at 2, 24, and 8 h, respectively (*P < 0.05). GM-CSF mRNA levels at 24 h were not different. Values are the means ± SEM of four experiments.

HBECs stimulated by 1% of CC increased mRNA expression of LIF (2 h), IL-1 (24 h), and IL-8 (8 h) (P < 0.05; Figure 4) similar to PM₁₀ (EHC-93) stimulation. IL-1 mRNA expression at 8 h and IL-8 mRNA expression at 24 h were also significantly increased (P < 0.05; data not shown). However, GM-CSF mRNA expression was not increased by CC stimulation at 24 h (Figure 4) or at any other time points. M-CSF, MCP-1, and IL-18 mRNA were constitutively expressed, and the other cytokines of the two template sets, including IL-6, were not induced by CC stimulation (data not shown).

View larger version (69K): [in this window] [in a new window]   Figure 4.   mRNA expression of LIF, GM-CSF, IL-1, and IL-8 after incubation in medium alone (control; open bars) and 1% CC (gray bars). GAPDH was used to correct for lane loading. Expression was quantified using densitometry. Results shown in the bar graph are the percentage change from control (medium alone) values at the time point of maximum cytokine mRNA expression in response to CC, which was 2, 24, and 8 h for LIF, IL-1, and IL-8, respectively. GM-CSF mRNA expression was not detected even after 24 h stimulation with 1% CC. Values are means ± SEM of three experiments. mRNA expression of LIF (2 h), IL-1 (24 h), and IL-8 (8 h) increased significantly compared with control (**P < 0.05).

Effect of PM₁₀ on Release of Cytokine Protein

Figure 5 shows cytokine levels in supernatants of HBECs incubated with medium alone (control), with 100 and 500 µg/ml of PM₁₀ suspension (EHC-93), and with 1 µg/ml of LPS. Cytokine production by HBECs stimulated by 100 and 500 µg/ml of PM₁₀ suspension increased in a dose- dependent manner for LIF, GM-CSF, IL-1, and IL-8. Incubation of HBECs with 1 µg/ml of LPS did not significantly increase their cytokine production. The soluble fraction of 100 µg/ml PM₁₀ did not stimulate cytokine production in HBEC (LIF: 54.0 pg/ml; GM-CSF: 17.7 pg/ml; IL-1: 8.2 pg/ml, IL-8: 394.8 pg/ml) inasmuch as levels were similar to those of controls and, in the case of GM-CSF, IL-1, and IL-8, also after stimulation of 100 µg/ml of PM₁₀ suspension.

View larger version (22K): [in this window] [in a new window]   Figure 5.   The concentration of cytokines measured by ELISA in supernatants of the HBEC culture medium after 24 h incubation with medium alone (control, white bars), 100 µg/ml of PM10 suspension (striped bars), 500 µg/ml of PM10 suspension (black bars), and 1 µg/ml of LPS (gray bars). Cytokine concentrations of LIF, GM-CSF, IL-1, and IL-8 are expressed as picograms per milliliter. Values are means ± SEM of four experiments. *P < 0.01, **P < 0.05 compared with control.

	Discussion

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This study demonstrates that PM₁₀ collected in an urban environment (EHC-93) induced mRNA expression of IL-1, IL-8, GM-CSF, and LIF in HBECs in a dose-dependent manner. This increase in mRNA was accompanied by an increase in cytokine protein production. Inert carbon particles induced mRNA expression of IL-1, IL-8, and LIF. The data also show that the soluble fraction of the EHC-93 particles has a much smaller stimulating effect on the HBEC than does the whole particle. The cytokines produced by HBECs after exposure to PM₁₀ are capable of contributing to the local inflammatory response in the lung. Further, the release of these cytokines into the circulation could induce a systemic response that includes a stimulation of the bone marrow to release leukocytes and platelets (4, 5, 16). We postulate that both these local and systemic inflammatory responses contribute to the morbidity and mortality associated with exposure to these atmospheric particles.

The increases in IL-1, IL-8, and GM-CSF mRNA expression observed in this study are similar to those described in previous reports (10, 17). However the earlier reports were based on transformed bronchial epithelial cell lines (i.e., BEAS-2B, BET-1A), whereas we used freshly harvested primary bronchial epithelial cells to more closely represent the response of the human airway lining to PM₁₀ exposure. Most of the previous in vitro studies used generated diesel exhaust particles (DEP) or residual oil fly ash that are chemically and physically dissimilar from the particles we used, which were ambient air pollution particles collected over a major North American city (EHC-93). We did not observe an increase in mRNA levels of IL-6 and TNF- or the chemokines RANTES and MCP-1 that have been reported by other workers using transformed cells line (10, 11, 18). The influence of the type of particle was supported by the observation that inert carbon particles caused an increase in mRNA of IL-1, IL-8, and LIF (Figure 4) but not of GM-CSF. Taken together, these studies suggest that both the cell type and particle type are probably responsible for the discrepancies between our results and those of previous reports.

PM₁₀ is a complex mixture of organic and inorganic compounds, and the individual components responsible for their adverse health effects have not been identified. Bioavailable metals, biogenic and organic components, and the physical characteristics of particles have each been proposed to be important in the PM₁₀-induced inflammatory response in the lung (19, 20). Several workers recently suggested that the toxicity of PM₁₀ resides in their associated metals, such as iron (19), vanadium (10), or copper (11), because of their redox potential. Adamson and colleagues (20) reported that the soluble fraction of urban air particulate sample instilled into mouse lung induced an inflammatory response and lung injury similar to that of instilling the whole dust sample. They postulated that the metal ions in the soluble fraction of the PM₁₀ are responsible for the lung injury. Bayram and associates (21) showed that the filtrate from DEP significantly increased the release of IL-8, GM-CSF, and soluble intercullar adhesion molecule-1 from bronchial epithelial cells and attenuated their ciliary beat frequency. Several groups have proposed that these effects are the result of compounds adsorbed onto the carbonaceous core of DEP, rather than the particles themselves (17, 21). Murphy and colleagues (22) reported that the bioreactivity of carbon black is related to particle size and surface area as well as to the surface chemistry. Studies from Boland and colleagues suggested that the organic compounds of DEP are more likely to induce the cytokine response in epithelial cells rather than the carbonaceous core because carbon black, activated charcoal, or graphite had no effect on cytokine release from bronchial epithelial cells (17). The ambient PM₁₀ (EHC-93) used in this study is a complex aggregate of elemental and organic carbons, metals, sulfates, nitrates, and organic contaminants (12, 14, 20), and the soluble metal components in these particles have been proposed as the major contributor to their ability to stimulate AM to produce cytokines (23). Our study shows that the soluble compounds from the particles (EHC-93) have a smaller effect on cytokine production by the bronchial epithelial cells than do the particles themselves. These data were supported by our experiments using small inert carbon particles without endotoxin, showing their ability to induce mRNA expression of LIF, IL-1, and IL-8 in HBECs.

The particles we used contained trace amounts of endotoxin. Becker and colleagues have suggested that endotoxin contributes to the cytokine production by AM exposed to EHC-93 because cytokine production was partly inhibitable by polymyxin B (12). Trace amounts of endotoxin (~ 3.0 ng/ml in 100 µg/ml of PM₁₀ suspension) were detected on the EHC-93 particles used in this study. In our study, a much larger dose of LPS (1 µg/ml) had a much smaller effect on HBECs (only IL-1 and GM-CSF were increased), suggesting that the endotoxin contamination of the PM₁₀ (~ 100 times less) was not responsible for the mRNA induction. Further, instillation of a similar amount of endotoxin into the lungs of rabbits failed to stimulate the bone marrow (16). Therefore, we conclude that endotoxin was not a critical factor in HBEC stimulation and suggest that the endotoxin component of the EHC-93 cannot explain the cytokine production by HBECs exposed to EHC-93.

The finding that PM₁₀ induced LIF mRNA expression and protein release from HBECs is, to our knowledge, a novel observation. LIF, a multifunctional glycoprotein that is related to the IL-6 family of cytokines, displays biologic activities ranging from the differentiation of myeloid leukemic cells into a macrophage lineage, effects on bone metabolism, inflammation, neural development, embryogenesis, and the maintenance of implantation (24). LIF by itself does not possess colony-stimulating activity when incubated with early human bone-marrow cells expressing the cell surface marker CD34. However, it is an important cofactor for the colony formation induced by IL-3 and is as potent as G-CSF and IL-6 (25). This suggests that LIF may have a role in the regulation of immature hematopoietic cells. LIF is released from a variety of cell types in vitro, and Knight and colleagues have demonstrated that IL-1 induced the accumulation of LIF mRNA and protein release in cultures of human bronchial epithelial cells (26). This effect was similar in lung fibroblast and airway smooth-muscle cells, and these investigators proposed that LIF is an important cytokine in the regulation of the inflammatory response in the lung in disorders such as asthma and bronchitis. In addition to its proinflammatory response in the lung, we suspect that LIF may also contribute to the bone-marrow stimulation associated with PM₁₀ exposure.

Our general hypothesis is that cytokines produced in the lung after PM₁₀ exposure are released into the circulation, inducing a systemic inflammatory response, and that this systemic response contributes to the cardiopulmonary morbidity and mortality associated with PM₁₀ air pollution. We measured the cytokines known to contribute to this systemic inflammatory response, specifically those that have an effect on the hematopoietic system. Several studies have shown that granulocytes released from an activated bone marrow have an enhanced ability to damage tissues but an impaired ability to destroy invading pathogens (27). Previous studies from our laboratory have shown that immature polymorphonuclear leukocytes (PMNs) released from the bone marrow by acute pneumonia (6), endotoxemia (7), and cigarette smoke exposure (8) preferentially sequester in the pulmonary microvessels. These immature PMNs were also slow to migrate out of the capillaries into an inflammatory site (6, 7) and their activation in the lung microvessels could result in endothelial damage. An increased burden of immature PMNs in the circulation after PM₁₀ exposure (4, 16) might contribute to the observed decrease in lung function associated with chronic exposure to particulate air pollutants (2, 28).

IL-1, IL-8, and GM-CSF are all cytokines known to stimulate the bone marrow to release leukocytes (29) and the liver to produce acute phase proteins (30), and to activate endothelium (31). Tan and colleagues showed that during the forest fires in Southeast Asia in 1997, soldiers who performed exercises outdoors on a regular basis as part of their combat training demonstrated a systemic inflammatory response that included bone-marrow stimulation during the period of the haze which returned to normal after the haze cleared (5). In collaboration with Tan and colleagues, follow-up studies on serum samples from these subjects showed an increase in IL-1 and GM-CSF during the haze period, with decreased levels after the haze had cleared (unpublished data, Stephan F. Van Eeden). This suggests that these cytokines produced in the lung during acute exposure spill over into the circulation and elicit the systemic component of the inflammatory response. Our data show that human bronchial epithelial cells in culture respond to ambient urban particulate matter. It is not possible to extrapolate the magnitude of this response observed in our cell culture system to a response of the epithelial cells in vivo. Nevertheless, the fact that a dose-response pattern can be established in the cell culture model supports the interpretation that a qualitatively equivalent response could be initiated in situ from the bronchial epithelium during inhalation exposure to air pollutants and deposition of particles.

Cytokines produced by HBECs when exposed to PM₁₀ could also contribute to the local inflammatory response. IL-1 plays an important role in neutrophilic and eosinophilic inflammation by increasing the expression, synthesis, and release of several proinflammatory cytokines and cell adhesion molecules (13). IL-8 is one of the most potent activators and chemoattractants for neutrophils, and it contributes to neutrophilic inflammation in chronic bronchitis and emphysema (32). It has also been shown to be a potent chemotactic factor for eosinophils and T lymphocytes as well, and therefore may have a role in the pathophysiology of allergic airway disease (33). GM-CSF, crucially important in stimulating the turnover and release of granulocytes and macrophages from the bone marrow, has recently been shown to be a potent neutrophil degranulation factor (34). It also prolongs the survival of neutrophils and eosinophils in a local inflammatory environment (35). Taken together, these cytokines could promote the local inflammatory response in the lung after PM₁₀ exposure, enhance the sensitivity of asthmatic individuals to air pollutants, and contribute to the higher rates of lung function decline associated with exposure to high levels of PM₁₀ air pollution (3).

In summary, the data reported here show that exposure of primary HBECs to urban air pollution particles increase mRNA expression and release of IL-1, IL-8, GM-CSF, and LIF. These cytokines will enhance the development of airway disease by enhancing the local inflammatory response in the airways and could also contribute to the systemic component of this inflammatory process.