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Published ahead of print on November 20, 2003, doi:10.1165/rcmb.2003-0123OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 744-750, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0123OC

Particle–Epithelial Interaction

Effect of Priming and Bystander Neutrophils on Interleukin-8 Release

Yaoyu Ning, Florence Tao, Guozhong Qin, Amy Imrich, Carroll-Ann Goldsmith, Zhiping Yang and Lester Kobzik

Department of Environmental Health, Harvard School of Public Health, and Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts

Address correspondence to: Dr. Lester Kobzik, Physiology Program, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail: lkobzik{at}hsph.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Exposure to ambient air pollution particles causes greater health effects in individuals with preexisting inflammatory lung diseases. To model inflammatory priming in vitro, HTB54 lung epithelial cells were pretreated with tumor necrosis factor-{alpha} (TNF-{alpha}) and then exposed to a panel of environmental particles, including concentrated ambient particles (CAPs). TNF-{alpha} priming significantly enhanced interleukin (IL)-8 secretion in response to CAPs and other urban air particles in HTB54 cells. Enhancement was seen with whole CAP suspensions as well as with its separate water-soluble and -insoluble components. Treating CAP suspensions with 20 µM deferoxamine or 2 mM dimethylthiourea attenuated the enhancement, indicating that transition metals and oxidative stress participate in the CAPs-dependent IL-8 response of primed cells. Because activated neutrophils are also present in diseased lungs and are sources of additional oxidative stress on epithelial cells, primed HTB54 cells were cocultured with activated neutrophils. Wild-type neutrophils markedly enhanced IL-8 release to CAPs in primed HTB54 cells, an effect substantially diminished when neutrophils from NADPH knockout mice were used. Cytokine priming and interactions with activated neutrophils can amplify lung epithelial inflammatory responses to ambient air particles.

Abbreviations: alveolar macrophages, AMs • bronchoalveolar lavage, BAL • concentrated ambient particles, CAPs • diethylenetriaminepenta-acetic acid, DETA • desferrioxamine, DFX • dimethylthiourea, DMTU • interleukin, IL • knockout, KO • lipopolysaccharide, LPS • macrophage inflammatory protein-2, MIP-2 • phosphate-buffered saline, PBS • polymorphonuclear neutrophils, PMNs • residual oil fly ash, ROFA • tumor necrosis factor-{alpha}, TNF-{alpha} • urban air particles, UAP • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Epidemiologic studies have reported a significant association between elevated levels of ambient air particles (with mean aerodynamic diameter of <= 2.5 µm) and increased respiratory and cardiovascular morbidity and mortality (1–3). One important biological mechanism of these health effects appears to be pulmonary inflammation triggered by particle exposure (4–6).

Lung epithelial cells participate in pulmonary inflammation by producing mediators such as cytokines and eicosanoids that recruit and activate hematopoetic inflammatory cells, and by causing adhesion-dependent activation and degranulation of these cells (reviewed in Ref. 7). In response to ambient air particles, airway epithelial cells increase nuclear factor-{kappa}B activity, secrete the cytokines interleukin (IL)-6 and IL-8, and increase surface expression of intercellular adhesion molecule 1 (ICAM-1) (8, 9). Airway epithelial-derived IL-8 elicits polymorphonuclear neutrophil (PMN) and eosinophil chemotaxis (10, 11), and likely mast cell chemotaxis as well (12).

Both epidemiologic data (1, 2) and experimental observations in animal models (13, 14) indicate that preexisting disease can increase susceptibility to particle health effects. We postulate that "priming" of lung epithelial cells by inflammatory mediators or cytokines amplifies proinflammatory responses to ambient air particles. Moreover, the alveolar milieu of diseased lungs includes activated inflammatory cells (e.g., PMNs) which release oxidants that might further amplify epithelial responses to particles. In a previous study, the lung epithelial cell line A549 produced significantly greater amounts of IL-8 in response to the experimental particles {alpha}-quartz and residual oil fly ash (ROFA) after priming with tumor necrosis factor-{alpha} (TNF-{alpha}) (15). The present study sought to expand these observations by using a panel of "real world" particles (concentrated ambient air particles [CAPs]) collected on different days. CAPs are composed of the anthropogenic particles that are most closely associated with health effects (size restricted to <= 2.5 µm diameter) and can be collected in real time (16). We tested the effect of TNF-{alpha} priming on CAPs-stimulated IL-8 secretion from the human bronchial epithelial cell line HTB54. The role of oxidant-dependent mechanisms in these responses was evaluated by using antioxidants, by testing the effects of enzymatically generated exogenous oxidants, and by coculturing with PMNs.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Animals
Female CD rats (12–16 wk old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). NADPH oxidase–deficient mice (B6.129S6-Cybbtm1Din [17]) or control C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). All animal experimentation was conducted under protocols approved by an institutional review board.

Buffers and Reagents
Phosphate-buffered saline (PBS), all cell culture media, and the Chromogenic Limulus Amebocyte Lysate kit for testing endotoxin were purchased from BioWhittaker (Walkersville, MD). Fetal bovine serum was purchased from Gemini Bio-Products (Calabasas, CA). Recombinant human TNF-{alpha} was purchased from Endogen (Woburn, MA). Dimethylthiourea (DMTU), desferoxamine (DFX), and diethylenetriaminepenta-acetic acid (DETA) were purchased from Sigma Chemical Co. (St. Louis, MO).

Particles and Particle Suspension Preparation
CAPs of respiratory size (<= 2.5 um) were collected from ambient Boston air onto teflo filters (Teflo, 47 mm diameter, 2 µm pore size; Gelman Sciences, Ann Arbor, MI) by the Harvard concentrator (16). Filters were weighed before and after particle collection and the total particle mass on each filter was calculated. The samples used in this study were collected on individual days from all four seasons during 1997–1998. To prepare particle suspensions, CAPs filters were cut into small strips, immersed in sterile endotoxin-free 0.9% sodium chloride, bath sonicated for 3 min, then probe sonicated three times for 1 min each time (Ultrasonics, Plainview, NY). The filter strips were removed, 100 µl of the particle suspension was weighed, and the mass amount released from each filter was calculated. The concentrations of particle suspensions obtained from individual CAPs filters were subsequently calculated. Aliquots of CAPs suspensions were stored frozen (–20°C) and were briefly bath sonicated before use (Laboratory Supplies Inc., Hicksville, NY).

The urban air particle SRM 1649 (UAP) was collected from ambient Washington D.C. air and is a Standard Reference Material purchased from the National Bureau of Standards (Washington, D.C.). ROFA and titanium dioxide (TiO2, mean diameter ~ 1 µm) were generously provided by Dr. J. Godleski and Dr. J. Brain, respectively (18, 19). Suspensions of UAP, ROFA, and TiO2 were prepared in sterile endotoxin-free 0.9% sodium chloride, stored frozen, and briefly sonicated before use.

In experiments to delineate the bioactive component(s) of CAPs, particle suspensions (referred to as whole suspension) were centrifuged at 13,000 x g for 20 min. The supernatant was removed (referred to as soluble component) and the pellet was resuspended in an equal volume of 0.9% sodium chloride (referred to as particle component).

Cell Lines and Cell Culture
Human lung epidermoid carcinoma Calu-1 cells (HTB54) were obtained from ATCC (Manassas, VA) and grown in McCoy's 5a medium supplemented with 10% non–heat-inactivated fetal bovine serum. Cells were seeded at 2 x 106 per 100 x 20 mm dish (Corning Inc., Corning, NY) and grown for 48 h at 37°C to obtain subconfluent cultures for all experiments.

Rat alveolar macrophages (AMs) were isolated by bronchoalveolar lavage (BAL) with PBS and prepared for in vitro use as previously described (14). Murine elicited neutrophils were obtained by intraperitoneal injection of 1 ml 3% thioglycollate and collected by peritoneal lavage 4 h later. The cells were resuspended in PBS after centrifugation, and determined to be > 95% PMNs by microscopic evaluation of Wright-Giemsa stained cytocentrifuge preparations. For coculture experiments, 5 x 104 PMNs were added per well just before addition of particles.

Cell Priming and Stimulation
HTB54 cells were seeded at 2.5 x 104/200 µl per well in 96-well plates (CoStar, Cambridge, MA). After 24 h (80% confluent), either fresh media or fresh media containing TNF-{alpha} (final concentration: 25, 5, 1, or 0.1 ng/ml) was added to the wells. The media was removed after another 24 h and the particles were added to stimulate the cells. In experiments investigating the role of particle oxidants in IL-8 responses, the particles were pre-incubated with antioxidants (DMTU and/or DFX) at room temperature (RT) for 10 min. After the cells were exposed to particles for 24 h, the supernatants were collected and stored at –70°C for subsequent IL-8 measurements.

Rat AMs were primed with 200 ng/ml lipopolysaccharide (LPS) for 3 h at 37°C as previously reported (14).

IL-8 Quantitation
IL-8 in culture medium samples was quantitated by enzyme-linked immunosorbent assay. Briefly, polystyrene 96-well plates (NalgeNunc International, Rochester, NY) were coated with 1 µg/ml mouse anti-human IL-8 (R&D Systems, Minneapolis, MN) overnight at room temperature. After blocking the plates with 1% bovine serum albumin (BSA; Sigma), the samples or standards were added. Captured IL-8 was detected with 4 µg/ml polyclonal rabbit anti-human IL-8 (Endogen, Woburn, MA) and the signal was amplified by adding 1:250 biotinylated goat-anti rabbit IgG (Vector, Burlingame, CA) followed by 1:250 ABC (Vector). The signal was developed with tetramethylbenzidine one step substrate (DAKO, Carpinteria, CA) and development was terminated with 2 N sulfuric acid. The plate was read from 450–550 nm on a microplate reader (Softmax; Molecular Devices, Menlo Park, CA) and the IL-8 concentration of each sample was calculated from the internal standard curve using Softmax software. Macrophage inflammatory protein-2 (MIP-2) was measured by enzyme-linked immunosorbent assay as previously reported (20).

Statistical Analyses
All data are reported as mean ± SEM. Comparisons were made by ANOVA with Fisher's PLSD adjustment to correct for multiple groups using the Statview software package (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
IL-8 Production by TNF-{alpha}–Primed HTB54 Cells in Response to ROFA, UAP, and TiO2
The effect of TNF-{alpha} priming on IL-8 production by HTB54 cells in response to particles was initially tested with the experimental particles ROFA, UAP, and TiO2. ROFA was collected from the boilers of power plants and so represents only a fraction of the types of respirable particles present in ambient air (18). UAP was collected from ambient air, but also contains larger particles of nonrespirable size, organic substances (most notably bacterial LPS and fungal spores) that elicit biological effects that may confound detecting the effects of specific respirable size ambient particles (21). Despite these limitations, however, both ROFA and UAP are useful as positive controls to assess the biological effects of pathogenic air pollution particles. TiO2 served as a negative control because it is typically biologically inert (15, 22). Figure 1A shows the effect of increasing TNF-{alpha} concentration on the IL-8 response of HTB54 cells exposed to 100 µg/ml of ROFA or UAP. Priming with TNF-{alpha} alone increased IL-8 secretion (open circles). Subsequent exposure to ROFA (filled squares) or UAP (filled circles) significantly augmented the IL-8 response in a TNF-{alpha} dose-dependent manner (P < 0.05 at 5 and 25 ng/ml TNF-{alpha}; ROFA or UAP versus control). These data show that 5 ng/ml TNF-{alpha} was sufficient to prime HTB54 cells for an augmented response to particles so this concentration was used in subsequent experiments.



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  		Figure 1. TNF-{alpha} priming enhances IL-8 release by the human lung epithelial cell line HTB54 in response to toxic (ROFA and UAP) and inert (TiO2) particles. Cells were primed with TNF-{alpha} for 24 h, then exposed to particles for another 24 h. (A) Dose–response analysis of TNF-{alpha} priming on IL-8 secretion by HTB54 cells exposed to 100 µg/ml ROFA (filled squares), UAP (filled circles), or no added particles (open circles) (*P < 0.05 versus control at 5 and 25 ng/ml TNF-{alpha} for both ROFA and UAP; n >= 4). (B) Dose–response analysis of IL-8 release by HTB54 cells in response to ROFA (filled squares, primed HTB54; open squares, unprimed), UAP (filled circles, primed; open circles, unprimed), or TiO2 (filled diamonds, primed; open diamonds, unprimed) after priming with TNF-{alpha} 5 ng/ml for 24 h. *P < 0.05 versus no particle control, all particle concentrations; **P < 0.05 versus control at 250 µg/ml only; #P < 0.05 versus no particle control at 100, 250 µg/ml; n >= 6.

 
Figure 1B shows the IL-8 response to increasing particle dose in HTB54 cells unprimed (open symbols) or primed with 5 ng/ml TNF-{alpha} (filled symbols). No significant effects on cell viability were seen at the concentrations reported here (> 90% viability, data not shown). Higher concentrations (> 250 µg/ml for ROFA and UAP) diminished viability (< 80% and 70%, respectively), so these were excluded from the analyses. In unprimed cells, 100 and 250 µg/ml ROFA and UAP stimulated significant responses above baseline (P < 0.05). Priming increased the sensitivity of the cells to ROFA and UAP because 50–250 µg/ml of these particles were able to stimulate responses significantly higher than basal (P < 0.05). Priming also increased the magnitude of IL-8 responses to ROFA and UAP by > 10-fold at every particle concentration. TiO2 did not elicit IL-8 responses in unprimed cells, but did stimulate a significant, albeit modest, signal in primed cells at the highest concentration tested (250 µg/ml). Priming with 5 ng/ml TNF-{alpha} therefore shifted the IL-8 dose response to particles leftward and upward.

IL-8 Production by TNF-{alpha}–Primed HTB54 Cells in Response to CAPs
TNF-{alpha} priming of HTB54 cells similarly enhanced the IL-8 response to CAPs. Figure 2A shows the IL-8 secreted from unprimed (open circles) and 5 ng/ml TNF-{alpha}–primed cells in response to increasing concentrations of CAPs. CAPs at < 300 µg/ml had no significant effect on cell viability (data not shown). The data are pooled from five experiments each using CAPs samples collected on five different days. None of the CAPs concentrations stimulated an IL-8 response greater than baseline in unprimed cells. In primed cells, 50–300 µg/ml CAPs stimulated significant IL-8 responses (P < 0.05). However, there was considerable variability in the CAPs responses of primed cells due to variable potency of the different CAPs samples. Figure 2B shows IL-8 responses to the five different CAPs samples in primed cells (pooled for Figure 2A).



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  		Figure 2. TNF-{alpha} priming enhances IL-8 release by HTB54 cells in response to CAPs collected on five different days. Cells were primed for 24 h with 5 ng/ml TNF-{alpha}, then exposed to different CAPs samples. (A) Summary dose–response analysis of IL-8 release by HTB54 cells in response to all CAPs samples (filled circles, primed HTB54; open circles, unprimed). *P < 0.05 versus no particle control for 50–300 µg/ml CAPs, n = 5. (B) Variability of IL-8 release by primed HTB54 cells in response to five individual CAPs samples (pooled data shown in A).

 
Contribution of Soluble and Insoluble CAPs Components to Chemokine Production
To determine whether amplified IL-8 production was associated with the soluble or insoluble components of CAPs, unprimed and TNF-{alpha}–primed HTB54 cells were incubated with the whole suspension (whole, 100 µg/ml), the insoluble resuspended particle (insoluble), or the water-soluble (soluble) component of CAPs for 24 h. As shown in Figure 3A, unprimed cells had no IL-8 response to any of the CAPs components. Primed cells secreted significantly higher IL-8 in response to whole, insoluble, or soluble components of CAPs (Figure 3A). However, the whole and soluble components elicited significantly higher responses than the insoluble component (P < 0.05).



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  		Figure 3. Role of water-soluble and -insoluble CAPs components in enhanced IL-8 release. CAPs were fractionated into soluble and insoluble fractions by centrifugation. (A) IL-8 production by unprimed and TNF-{alpha}–primed (5 ng/ml, 24 h) HTB54 cells after 24 h incubation with the whole suspension (black bars), water-soluble (light gray bars), or water-insoluble (charcoal gray bars) components of CAPs. Control samples contained cells and media only (white bars). In primed HTB54 cells, both the soluble and insoluble components of CAPs suspensions increased IL-8 release (*P < 0.05, n = 8). A subset of CAPs samples (n = 3) was used to compare HTB54 and AM responses. (B) Both soluble and insoluble components increased IL-8 release by primed HTB54 epithelial cells (*P < 0.05 versus no particle control, n = 3). (C) Only the insoluble particulate fraction increased MIP-2 release in rat AMs (AMs primed with LPS 200 ng/ml, 3 h; *P < 0.05 versus whole suspension or insoluble particulate–treated groups).

 
These data contrasted with previous observations in a different lung cell, the AM, in which the insoluble but not soluble component of CAPs stimulated LPS-primed rat AMs to secrete MIP-2 (a rodent CXC chemokine related to IL-8 [23]) (20). To investigate this cell type–dependent difference, IL-8 responses in HTB54 cells were compared with MIP-2 responses in rat AMs after stimulation with the same CAPs components. A subset of the CAPs samples used in Figure 3A was fractionated and tested again on primed HTB54 cells and rat AMs. IL-8 responses in TNF-{alpha}–primed HTB54 cells were again significantly increased by the whole suspensions, insoluble, and soluble components of these CAPs samples (Figure 3B). In LPS-primed AMs, however, only the whole suspensions and insoluble components stimulated a MIP-2 response (Figure 3C).

Role of CAPs Transition Metals and Oxidants on Amplified IL-8 Production
In studies with ROFA and UAP, epithelial inflammatory responses appeared to be associated with oxidative stress exerted by transition metals in the particles (9, 24). To examine the role of CAPs transition metals in the amplified IL-8 response of TNF-{alpha}–primed HTB54 cells, CAPs samples were treated with 20 µM DFX (to chelate iron and other transition metals [25]) before incubating with unprimed or primed cells. Similarly, to examine the role of oxidants in the amplified IL-8 response, CAPs samples from the same batch were treated with 2 mM DMTU (to scavenge oxygen free-radicals [24]) before incubating with unprimed or primed cells. Figure 4 shows the effects of DFX and DMTU on IL-8 responses to 100 µg/ml CAPs in unprimed and primed HTB54 cells. Unprimed cells did not increase IL-8 in response to CAPs, so DFX or DMTU treatment had no measurable effect. Basal IL-8 levels of primed cells was unaffected by exposure to DFX or DMTU. However, both DFX and DMTU significantly attenuated the IL-8 response to CAPs in primed cells (~ 70% inhibition, P < 0.05), suggesting that transition metals and oxidative stress are involved in CAPs-dependent IL-8 production. The two agents together did not reduce the CAPs-induced IL-8 signal to any greater extent than either agent alone (n = 5, data not shown). Similarly, combined DMTU and DFX pretreatment of ROFA had no greater effect than either agent alone on IL-8 responses in primed HTB54 cells (n = 2, data not shown).



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  		Figure 4. Effect of antioxidants on IL-8 production by untreated and TNF-{alpha}–primed HTB54 cells exposed to CAPs. HTB54 cells were incubated with CAPs (n = 5) ± vehicle (black bars), DMTU 2 mM (white bars) or DFX 20 µM (gray bars) for 24 h. In primed HTB54 cells, DMTU or DFX significantly inhibited the increased IL-8 release by CAPs. *P < 0.05 versus 100 µg/ml CAPs alone, n = 5.

 
Effect of Exogenous Oxidants on IL-8 Responses
To investigate the effect of additional oxidative stress on IL-8 responses, the HTB54 cells were exposed to glucose oxidase during the 24 h incubation with CAPs. Glucose oxidase generates a steady flux of hydrogen peroxide (H2O2) from glucose, water, and oxygen in the culture media (26). The exogenous H2O2 generated by glucose oxidase had no effect on IL-8 in unstimulated, unprimed cells but augmented the IL-8 response in CAPs-stimulated unprimed cells (Figure 5). Similarly, exogenous H2O2 did not affect the increased basal IL-8 levels due to TNF-{alpha} priming alone in a statistically significant manner (P = 0.28), but augmented the IL-8 response in CAPs-stimulated primed cells in a dose-dependent manner (P < 0.05). The data suggest that exogenous oxidants could further potentiate CAPs-mediated IL-8 release by lung epithelial cells.



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  		Figure 5. Effect of exogenous oxidants on IL-8 release by HTB54 cells. A steady-state flux of exogenous H2O2 was generated during the 24-h 100 µg/ml CAPs exposure by adding glucose oxidase to the culture media at the outset (white bars: control; black bars: 2.5 µM/h H2O2; gray bars: 10 µM/h H2O2). H2O2 increased IL-8 release by HTB54 cells, particularly in TNF-{alpha}–primed cells exposed to CAPs. *P < 0.05 versus control, n = 5.

 
Effect of Coculture with PMNs on IL-8 Responses
A potential source of exogenous oxidants within the alveolar milieu in vivo could be the PMNs present in diseased lungs (27). To test whether PMNs could affect IL-8 responses of HTB54 cells to CAPs, unprimed or primed HTB54 cells were cocultured with PMNs, and the cocultures were subsequently exposed to 100 µg/ml CAPs. The PMNs were used with or without activation by 12-phorbol 13-myristate acetate (PMA; 0.1 µM for 5 min), a treatment known to induce a respiratory burst and oxidant release (28). Figure 6A shows that unactivated PMNs did not affect basal or CAPs-dependent IL-8 responses of unprimed or primed HTB54. Coculture with PMA-activated PMNs markedly elevated IL-8 in unprimed and primed HTB54 cells (Figure 6B), shifting the dose–response curves leftward and upward. Thus, interaction with activated PMNs potentiated basal and CAPs-dependent IL-8 responses of HTB54 cells.



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  		Figure 6. IL-8 release by HTB54 cells is augmented by coculturing with activated PMNs. Elicited PMNs (5 x 104/well) were introduced to HTB54 cell cultures immediately before 100 µg/ml CAPs exposure. (A) Increasing the number of PMNs to HTB54 cultures had minimal effects on IL-8 release (open circles, unprimed, no particles; filled squares, unprimed +CAPs; open diamonds, primed, no particles; filled circles, primed +CAPs). (B) PMA-treated (100 nM, 5 min) PMNs substantially increased IL-8 release by HTB54 cells, particularly in primed cells incubated with CAPs. *P < 0.01 versus other groups; n >= 4, except n = 1 with 105 PMNs.

 
Regulation of IL-8 Responses by PMN NADPH Oxidase
The NADPH oxidase complex is the major source of oxidants released by PMNs during a respiratory burst, such as induced by PMA (29). To determine the role of PMN NADPH oxidase in PMN potentiation of IL-8 responses in HTB54 cells, IL-8 was compared from supernatants of HTB54 cells cocultured with PMA-activated PMNs (50 x 103/well) obtained from wild-type (WT) or NADPH oxidase knockout (KO) mice. Figure 7 shows that coculture of unprimed or primed HTB54 cells with WT PMNs potentiated the basal and CAPs-stimulated (100 µg/ml) IL-8 responses as seen in Figure 6B. The effects seen with WT PMNs on both basal and CAPs-mediated IL-8 release were substantially diminished when KO PMNs were used. Absence of NADPH oxidase diminished substantially (but did not eliminate) the ability of activated PMNS to further augment IL-8 release by primed lung epithelial cells.



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  		Figure 7. PMN NADPH oxidase augments CAPs-mediated IL-8 release in HTB54 cells. HTB54 cells cocultured with PMA-stimulated PMNs (5 x 104/well) from wild-type or NADPH-oxidase deficient mice were exposed to 100 µg/ml CAPs for 24 h. NADPH oxidase deficiency reduced the ability of PMNs to augment IL-8 release (white bars: control; black bars: WT PMNs; gray bars: KO PMNs). *P < 0.05 versus control or KO PMNs, n = 5.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
The data demonstrate that primed epithelial cells have augmented proinflammatory responses to CAPs in vitro. Fractionation of CAPs showed that soluble and, to a lesser degree, insoluble components caused increased IL-8 release. Antioxidant and metal chelator inhibitors blocked the response to CAPs, consistent with metal-dependent oxidant mechanisms. Additional oxidative stress imposed by other sources, such as activated PMNs, further enhanced the proinflammatory response to CAPs.

These findings support the epidemiologic observations that ambient particle exposure causes greater morbidity in people with preexisting disease (1, 3). They are also consistent with previous in vitro observations showing that LPS-primed AMs secrete higher levels of cytokines in response to CAPs than unprimed AMs (20). Collectively, the data suggest that ambient particles promote feedforward activation of alveolar cells that increases the severity of a preexisting condition. For example, when TNF-{alpha}–primed epithelial cells interacted with PMA-activated PMNs (reflective of the priming/activation extant in preexisting inflammatory conditions), a small number of PMNs (PMN:HTB54 ratio ~ 1:2) enhanced IL-8 secretion in response to CAPs. This enhanced IL-8 could lead to more PMN recruitment, more epithelial–PMN interaction (resulting in a downward spiral of an increasingly oxidative alveolar milieu), more cytokine secretion, and the potential for lung injury and physiologic impairment. It is relevant to note that CAPs exposure of healthy and diseased animals or humans in vivo has been associated with lung neutrophilia measured by BAL (6, 13, 30). Our data suggest a mechanism for this enhanced neutrophilia and argue that the activation state of cells before CAPs exposure strongly influences the ensuing inflammatory responses.

There is, however, evidence that CAPs exposure in vivo has inconsistent effects on neutrophilia as well. In a bronchitic rat model developed by Clarke and coworkers, CAPs exposure increased PMNs in the BAL (13). In another bronchitic rat model developed by Kodavanti and colleagues, CAPs exposure sometimes increased and sometimes had no effect on PMN numbers in BAL (31). We observed considerable variability in IL-8 responses of primed HTB54 cells to CAPs collected on different days. This was reminiscent of the variability in CAPs-triggered oxidative burst of AMs that tracked the daily variability in CAPs composition (31, 32). Factor analysis of CAPs components has revealed that certain biological responses were associated with certain components of CAPs, for example, BAL PMN percentage with the aluminum/silica factor (30). The inconsistent effects reported on neutrophilia in bronchitic rats in vivo may have therefore arisen from variable composition of the different CAPs samples (determined by temporal, climatic, and geographical heterogeneity) used in each study.

Fractionating the CAPs into water-soluble and -insoluble compartments revealed that IL-8 active components for TNF-{alpha}–primed HTB54 cells were present in both compartments. Similarly, in epithelial cells, Ghio and coworkers reported that soluble and insoluble compartments of ambient air particles stimulated IL-8 release from the (unprimed) human bronchial cell line BEAS-2B (33). In contrast, only the insoluble compartment was MIP-2 active for LPS-primed AMs in this study and in prior work from our laboratory (20). The basis for this intriguing cell-specific difference in response remains to be investigated.

Treating CAPs with DMTU or DFX substantially attenuated (~ 70%) the augmented IL-8 response to CAPs in TNF-{alpha}–primed HTB54 cells. Treating CAPs with both DMTU and DFX had no further effect, indicating that the transition metals and oxidative stress acted through similar mechanisms to augment IL-8 responses. Indeed, it is likely that the oxidative stress was metal-dependent, as other studies of CAPs samples have found that their oxidative capacity is primarily due to the presence of transition metals (34). Because unprimed HTB54 cells were unresponsive to CAPs, it appears that priming with TNF-{alpha} sensitized the cells to oxidative stress by transition metals in the particles. The cellular mechanisms of this increased sensitivity are unclear. Both water-soluble and -insoluble compartments of our CAPs samples contain oxidant activity when tested in a cell-free assay using oxidation of nonfluorescent dichlorofluorescin to a fluorescent reporter (A. Imrich and colleagues, unpublished observations). Moreover, transition metals and oxidant activity have been demonstrated to exist in both compartments of PM (33), so it is likely that both soluble and insoluble activities of CAPs were associated with metal-dependent oxidative stress. Iron is the most likely candidate metal, because it is present in CAPs and is strongly chelated by DFX (20, 34). However, additional studies are required to investigate the potential of other metals (e.g., Cu, Ni, Zn) to also contribute to epithelial cell responses in our system.

Neutrophils are likely to be present in the alveolar milieu of diseased lungs and have been observed after CAPs exposure in vivo (6, 13, 30). We postulated that in the presence of epithelial cells and CAPs, PMNs may produce O2- and H2O2 that would contribute additional oxidative stress and thereby amplify the epithelial IL-8 responses to CAPs. In proof-of-principle studies, exogenous H2O2 augmented IL-8 responses in both unprimed and primed HTB54 cells. More importantly, experiments using activated PMNs also demonstrated augmented responses to CAPs. The greatest effect was seen when a coculture of primed HTB54 cells and preactivated PMNs was exposed to CAPs. These experiments confirmed that interaction between epithelial cells and PMNs amplifies inflammatory responses to CAPs. Parallel observations that epithelial interaction with AMs also results in amplified inflammatory responses to particles (35) highlight the importance of intercellular communication in determining the intensity of biological responses to particles.

Similar results were seen using human and rat PMNs (data not shown), although we focused on experiments that allowed comparison of normal and NADPH oxidase-deficient murine PMNs. Coculturing HTB54 cells with preactivated PMNs obtained from NADPH oxidase knockout mice showed that the augmented basal IL-8 in unprimed and primed HTB54 cells from coculturing with PMNs was dependent on NADPH oxidase activity. Neutrophil NADPH oxidase also contributed in substantial part to (but did not explain all) the amplified IL-8 response of primed HTB54 cells cocultured with preactivated PMNs and exposed to CAPs.

In summary, cytokine priming and interactions with activated neutrophils can amplify lung epithelial inflammatory responses to ambient air particles. Oxidant constituents (e.g., soluble metals) of air particles are an important component. In addition, PMNs can amplify CAPs-stimulated epithelial-mediated inflammation by compounding oxidative stress and through additional mediator(s) that remain to be identified. In vitro studies using a coculture approach to model the alveolar milieu may provide insight into the complex interactions that lead to air particle toxicity.


   Acknowledgments
 
This research was supported by the NIH grants ES008129 and ES00002. Additional support was provided by the EPA grant R827353.

Received in original form April 2, 2003

Received in final form November 19, 2003


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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