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Published ahead of print on September 18, 2003, doi:10.1165/rcmb.2002-0273OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 597-604, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2002-0273OC

Oxidant-Injured Airway Epithelial Cells Upregulate Thioredoxin but Do Not Produce Interleukin-8

Karen L. Oslund, Lisa A. Miller, Jodie L. Usachenko, Nancy K. Tyler, Reen Wu and Dallas M. Hyde

Center for Comparative Respiratory Biology and Medicine, and Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California Davis, Davis, California

Address correspondence to: Dallas M. Hyde, Ph.D., The Center for Comparative Respiratory Biology and Medicine, School of Veterinary Medicine, One Shields Ave., Davis, CA 95616. E-mail: dmhyde{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We tested the hypothesis that oxidant-injured cells upregulate thioredoxin, whereas oxidant-stressed, but not injured, cells upregulate interleukin (IL)-8 after injury. We exposed primary human tracheobronchial epithelial cells and transformed human bronchial epithelial cells (BEAS-2B S.6) to 0, 200, 400, or 600 µM H2O2 for 1 h followed by an additional 7 h of incubation. Subsequently, the cells were double-labeled with markers of injury (either Ethidium Homodimer-1 for cellular injury or MitoTracker dye for functional mitochondria) or oxidant stress (5-[and 6]-chloromethyl-2',7'-dicholorodihydrofluorescein diacetate) and antibodies specific for the chemoattractants IL-8 or thioredoxin. We found significant inverse relationships between numbers and stained chemoattractant volumes of IL-8 and thioredoxin-positive cells with increasing H2O2 dose. Cells with mitochondrial injury produced thioredoxin but not IL-8, and oxidant-stressed cells were more likely to produce thioredoxin than IL-8. Isolated human neutrophils were more likely to colocalize with thioredoxin-positive BEAS-2B S.6 cells than thioredoxin-negative cells. The H2O2 injury did not induce significant apoptosis in the BEAS-2B S.6 cells as measured by caspase 3 activation. We conclude that oxidant-injured and stressed airway epithelial cells upregulate thioredoxin, but produce little IL-8, which may be important in airway epithelial cell–mediated multistep navigation of neutrophils to sites of oxidant injury.

Abbreviations: activator protein-1, AP-1 • bronchial epithelial growth medium, BEGM • bovine serum albumin, BSA • fluorescein isothiocyanate, FITC • Hanks' balanced salt solution, HBSS • interleukin, IL • nuclear factor IL-6, NF–IL-6 • nuclear factor-{kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • phycoerythrin, PE • room temperature, RT • tumor necrosis factor-{alpha}, TNF-{alpha} • tracheobronchial epithelial cells, TBE cells


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cellular injury and oxidative stress occur in numerous inflammatory conditions, but are often transient events as cells are repaired or removed. However, these events may have profound effects in orchestrating and enhancing the subsequent inflammatory and reparative responses. Numerous studies have shown that reactive oxygen species are important in activating transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B), nuclear factor–interleukin (IL)-6 (NF–IL-6), and activator protein-1 (AP-1), which in turn activate many critical genes involved in inflammation, including many cytokine genes. Cytokines, such as IL-8, are important in recruiting inflammatory cells to a site of injury that is followed by cellular repair. In a series of experiments, Hyde and colleagues have shown that the neutrophil influx following IL-8 production is critical in hastening airway epithelial repair after ozone exposure (1, 2).

Thioredoxin, a ubiquitous stress-inducible protein, has both intracellular and extracellular functions. Intracellularly, it maintains a cell's reduction/oxidation status by a disulfide bond within the conserved active site, Cys-Gly-Pro-Cys (3). Cellular redox control is critical for numerous cell functions including DNA binding activity of NF-{kappa}B and AP-1. Extracellular functions of thioredoxin include inhibiting apoptosis, autocrine growth factor effects on HTLV-1 or EBV-infected T cells and a promotion of B cell differentiation (4).

Recently, thioredoxin has been identified as a unique chemoattractant for human neutrophils, monocytes, and T cells. Similar to cytokines, thioredoxin can be released by certain cells, such as lymphocytes, upon activation. In contrast to typical chemokines, thioredoxin does not increase intracellular calcium in leukocytes and its action is G-protein–independent. Thioredoxin likely does not act through a chemokine receptor, but rather may initiate signal transduction for chemotaxis by oxidizing and cross-linking certain cell surface molecules (5). Functioning as a chemokine, thioredoxin is partially responsible for neutrophil migration across airway epithelium in an in vitro model (6). In this same study, neutrophil transepithelial migration was independent of IL-8.

Numerous studies have shown that tissues or populations of cells upregulate thioredoxin after oxidant stress. Stressors, including hydrogen peroxide (H2O2), ultraviolet irradiation, and tumor necrosis factor-{alpha} (TNF-{alpha}), upregulate thioredoxin production and release from a variety of cells (7, 8). Increased circulating levels of thioredoxin have been found in patients with rheumatoid arthritis (9) and HIV infection (10). The increase in circulating thioredoxin is likely associated with the inflammation and oxidative stress of these diseases. In the lung, thioredoxin mRNA is upregulated within 24 h after combined ozone and nitrogen dioxide exposure in rats (11), and gene expression is transcriptionally upregulated by retinol in the conducting airway epithelium of rhesus monkeys (12).

Thioredoxin can function as a link between oxidant stress and initiation of an inflammatory response. Once secreted, very low concentrations of thioredoxin (1–3 µM) strongly stimulate the cytokines TNF-{alpha}, IL-1, IL-2, IL-6, and IL-8 in several cell types (13). Establishing a direct interaction between thioredoxin and IL-8 upregulation, researchers have found thioredoxin production activates NF-{kappa}B, which in turn upregulates the IL-8 gene promoter after either retinoic acid or TNF-{alpha} treatment in airway epithelial cells (14).

To further understand the production and upregulation of thioredoxin and IL-8 at the cellular level, we investigated the relationship between oxidative stress and injury and cytokine production using airway epithelial cells exposed to H2O2. To our knowledge, this is the first paper to investigate whether individual oxidant-stressed or injured cells can produce IL-8 or thioredoxin. Thioredoxin is produced and upregulated by oxidant-stressed and injured airway epithelial cells in contrast to IL-8, which shows very limited production in injured or stressed cells. These results suggest that cytokine production is differentially regulated by cells depending on the type of stress or injury they have sustained during an oxidant insult.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Human tracheobronchial tissue was obtained from the Medical Center of the University of California, Davis (Davis, CA), Sacramento Organ Donation Foundation, Inc. (Sacramento, CA), and the National Disease Research Interchange (Philadelphia, PA). The University of California at Davis Human Subjects Review Committee approved all procedures involved in the tissue procurement. Primary tracheobronchial epithelial cell isolation and culture methods were performed as described previously (15). Transformed human bronchial epithelial cells (BEAS-2B S.6) were a gift from Dr. Curtis Harris (Bethesda, MD). The BEAS-2B S.6 cell line was derived from normal human bronchial epithelial cells immortalized with an SV40-adenovirus 12 hybrid virus (16). Both cell lines were maintained in serum-free bronchial epithelial growth medium (BEGM; Clonetics, San Diego, CA) without retinoic acid and trypsinized every week. Primary tracheobronchial epithelial cells (TBE) were used between passages 1 and 4. The BEAS-2B S.6 cells were used for experiments between passages 30 and 45, at which time they maintain transepithelial resistance (6).

Cell Injury and IL-8/Thioredoxin
A quantity of 2–3 x 104 TBE cells or BEAS-S.6 were plated and grown on LabTek four chamber slides (Nalge Nunc, Naperville, IL) until they reached confluence as determined by phase contrast microscopy. Before plating the TBE cells, the slides were coated with several drops of CellPrime (Cohesion, Palo Alto, CA) and dried overnight. For IL-8 staining, cells were stimulated for 24 h before the start of the experiment with TNF-{alpha} (Endogen, Woburn, MA) (10 ng/ml for the TBE cells and 20 ng/ml for the BEAS-2B S.6) and IL-1ß (Endogen) (10 units/ml for the TBE cells and 20 units/ml for the BEAS-2B S.6). Monolayers were exposed to 0, 200, 400, or 600 µM H2O2 in BEGM for 1 h at 37°C and 5% CO2 in the presence of 2 µM monensin (GolgiStop; Pharmingen, San Diego, CA). The media was removed, and monolayers were incubated for seven additional hours at 37°C with warmed BEGM and monensin (including TNF-{alpha}/IL-1ß for IL-8 experiments). Immediately afterwards, monolayers were incubated in the dark at 37°C with either 0.6 mM Ethidium Homodimer-1 (Molecular Probes, Eugene, OR) for 15 min for cellular injury or 125 nM MitoTracker Red CMXRos (Molecular Probes) for 20 min for mitochondrial injury. Monolayers were then fixed with 4% paraformaldehyde for 20 min at 4°C, and nonspecific staining was blocked with phosphate-buffered saline (PBS) plus 6% bovine serum albumin (BSA) for 30 min at room temperature (RT). Monolayers were permeabilized with 0.1% saponin in PBS+ 1% BSA and incubated overnight at 4°C with either fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-human IL-8 antibody (Pharmingen) or polyclonal goat anti-human thioredoxin antibody (American Diagnostica, Greenwich, CT). For the thioredoxin staining, an FITC-conjugated donkey anti-goat IgG secondary antibody (Chemicon, Temecula, CA) with 0.1% saponin in PBS+ 1% BSA was added to the monolayers for 45 min at RT. As a negative control, monolayers were stained with an FITC-conjugated mouse IgG2b,{kappa} isotype control antibody (Pharmingen) for IL-8 and the secondary antibody only or goat serum for thioredoxin.

Using stratified random sampling, the number of injured BEAS-2B S.6 (Ethidium Homodimer-1 positive or MitoTracker-negative) and IL-8–positive cells were counted on an epifluorescent microscope (Olympus Provis) in a x40 field (representing a 0.152 mm2 area per field) for 10 fields at each dose of H2O2. Additionally, using stratified random sampling four IL-8–positive cells were imaged at x100 in each of the 10 fields for a total of 40 IL-8–positive cells per H2O2 dose. The image of each cell was imported into Stereology Toolbox (Morphometrix, Davis, CA) for point counts to determine the volume of IL-8 per total cell volume (VIL-8/Vcell) (17). For thioredoxin staining of the BEAS-2B S.6 and all the TBE cell staining, 10 random images per H2O2 dose were taken at x40 on an epifluorescent microscope (Olympus Provis). Using Stereology Toolbox, the number of injured or uninjured IL-8 or thioredoxin-positive cells was counted (representing a 0.04 mm2 area per field for the BEAS-2B S.6 experiments and 0.0784 mm2 for the TBE cell experiments). Additionally, the volume of thioredoxin staining per total cell volume of four randomly selected thioredoxin-positive BEAS-2B S.6 cells were counted for each image (Vthioredoxin/Vcell) (17).

To assess the integrity of the Golgi apparatus after incubation with hydrogen peroxide, two control experiments were performed. First, monolayers of BEAS-2B S.6 cells were exposed to either 0 or 600 µM H2O2 in BEGM with TNF-{alpha}, IL-1ß, and monensin for 1 h at 37°C. This media was removed and the monolayers were incubated in BEGM with the cytokines and monensin for an additional 7 h. At this point, the monolayers were gently washed with Hanks' balanced salt solution (HBSS) and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing, the monolayers were incubated with 5 µM NBD C6-ceramide (Molecular Probes) in an ice bath for 30 min at 4°C for specific labeling of the Golgi apparatus. Monolayers were rinsed with HBSS containing 3.4 mg/ml BSA to remove excess ceramide, mounted, and examined under an epifluorescent microscope.

As a second control, monolayers of BEAS-2B S.6 were exposed to either 0 or 600 µM H2O2 in BEGM without cytokines or monensin. This media was removed and the monolayers were incubated in BEGM with 50 ng/ml TNF-{alpha}, 20 U/ml IL-1ß, and 2 µM monensin for an additional 7 h to induce maximal IL-8 stimulation. After this, the monolayers were fixed and stained for IL-8 as previously described. Using stratified random sampling, 10 fields were imaged with an epifluorescent microscope and the images imported into Stereology Toolbox. The number of IL-8–positive cells exposed to each dose of hydrogen peroxide was counted per mm2 surface area using unbiased stereologic methods (18).

Oxidant Stress and IL-8/Thioredoxin
A quantity of 1.5–3 x 105 TBE cells or BEAS-2B S.6 were grown to confluence on LabTek one chamber slides (Nalge Nunc, Naperville, IL). Before plating the TBE cells, the slides were coated with several drops of CellPrime. For IL-8 staining, cells were stimulated for 24 h before the start of the experiment with TNF-{alpha} and IL-1ß as previously described. Monolayers were loaded with 1 µM of the oxidant-sensitive dye, 5-(and 6)-chloromethyl-2',7'-dicholorodihydrofluorescein diacetate (Molecular Probes) in 3 ml BEGM and incubated for 15 min at 37°C and 5% CO2 in the dark. After washing, monolayers of BEAS-2B S.6 were exposed to various concentrations of H2O2 (0, 200, or 400 µM H2O2 for IL-8 or 200, 400, or 600 µM H2O2 for thioredoxin) in BEGM for 1 h at 37°C and 5% CO2 in the presence of 2 µM monensin (and TNF-{alpha}/IL-1ß for IL-8 experiments). Monolayers of TBE cells were exposed to 0 or 200 µM H2O2 (for IL-8) or 400 or 600 µM H2O2 (for thioredoxin) under similar conditions. An England slide finder (Electron Microscopy Sciences, Ft. Washington, PA) was glued to the bottom of the LabTek chamber with Nexaband (Veterinary Products Laboratories, Phoenix, AZ) to facilitate relocation of the cells. Using stratified random sampling, 20 fields of the live cells, at each H2O2 dose, were imaged using an epifluorescent microscope (Olympus Provis) with a x40 water immersion objective. Monolayers were kept between 4 and 21°C and covered in HBSS containing calcium and magnesium during the 25-min imaging. Immediately after imaging, monolayers were incubated with warmed BEGM plus monensin (and TNF-{alpha}/IL-1ß for the IL-8 experiments) for seven additional hours at 37°C and 5% CO2. Monolayers were fixed with 1% paraformaldehyde for 20 min at 4°C and nonspecific staining was blocked with PBS plus 6% BSA for 30 min at RT. Monolayers were permeabilized with 0.1% saponin in PBS+ 1% BSA and incubated overnight at 4°C with phycoerythrin (PE)-conjugated monoclonal mouse anti-human IL-8 antibody (Pharmingen) or polyclonal goat anti-human thioredoxin antibody. For thioredoxin staining, a rhodamine-conjugated donkey anti-goat IgG secondary antibody (Chemicon) with 0.1% saponin in PBS+ 1% BSA was added to the monolayers for 45 min at RT. As a negative control, monolayers were stained with PE-conjugated mouse IgG2b,{kappa} isotype control antibody (Pharmingen) for IL-8 or the secondary antibody only for thioredoxin. The next day, the same fields were relocated using the England slide finder and imaged at x40 on an epifluorescent microscope for IL-8 or thioredoxin positive cells. By comparing the two images, IL-8 or thioredoxin-positive/oxidant-stress–positive or –negative cells were counted using Stereology Toolbox. Additionally, oxidant stress positive and negative cells were counted for each H2O2 dose.

Identification of Apoptotic BEAS-2B S.6 Cells
Monolayers of BEAS-2B S.6 cells were plated on four chamber slides as previously described. The monolayers were incubated with either 0, 200, 400, or 600 µM H2O2 for 1 h at 37°C. The monolayers and sloughed BEAS-2B S.6 were stained for active caspase 3 both immediately after the 1-h incubation and following a 7-h postexposure incubation. To collect the sloughed cells, the BEGM and two washes with HBSS were collected and pooled. Cytospins were made from the collected media. Monolayers and cytospins were fixed with 4% paraformaldehyde for 20 min at 4°C. Nonspecific staining was blocked with PBS plus 6% BSA for 30 min at RT. Cells were stained with an FITC conjugate of anti-active caspase 3 (PharMingen) 1:100 in PBS + 1% immunohistochemical grade BSA (Vector, Burlingame, CA) overnight at 4°C in the dark. Cytospins of aged human neutrophils served as positive controls. Negative controls were incubated with PBS + BSA only. Slides were viewed on an epiflourescent microscope.

Colocalization of Human Neutrophils with Thioredoxin-Stained BEAS-2B S.6
Monolayers of BEAS-2B S.6 cells were plated on single-chambered slides as previously described. The monolayers were incubated with either 0 (control condition for nonspecific neutrophil binding) or 400 µM H2O2 for 1 h at 37°C. Human neutrophils were isolated from donor whole blood by an established Ficoll method (19). Isolated neutrophils were stained with a vital dye, PKH26 red (Sigma, St. Louis, MO) according to the manufacturer's instructions. Based on previous experiments in our laboratory, we estimated a 4:1 neutrophil to epithelial ratio for optimal cellular interaction and added 5.04 x 106 neutrophils to 3 ml of equilibrated BEGM (6). The neutrophils were added to the monolayers immediately following the 1 h incubation, and the co-culture was incubated for 1 h at 37°C. Nonadherent neutrophils were gently washed off the monolayers with PBS using a 25-gauge butterfly catheter at 30 cm water pressure for 1 min. The cells were fixed and stained for thioredoxin as previously described in this article. Each H2O2 concentration was performed twice.

Using stratified random sampling, 20 fields per co-culture were imaged at x40 on an epiflourescent microscope. Then, using Stereology Toolbox (Morphometrix, Davis, CA), the number of neutrophils associated with thioredoxin-positive and thioredoxin-negative cells were counted. Colocalization was defined as neutrophils touching or within one-half epithelial cell diameter of the thioredoxin-positive or -negative cells. When neutrophils were surrounded by multiple epithelial cells, only one BEAS-2B S.6 cell was counted with thioredoxin-positive cells taking precedence over thioredoxin-negative cells. The number of neutrophils associated with thioredoxin-positive or -negative cells was calculated as a percentage of neutrophil binding above baseline conditions (0 µM H2O2).

Statistical Analysis
To determine if there was a significant overall relationship between IL-8 and thioredoxin-positive BEAS-2B S6 cells over all the H2O2 concentrations, partial nonparametric correlations, Kendall's {tau}b statistic, and Spearman test were performed (SAS; SAS Institute Inc., Cary, NC). In addition, separate ANOVA tests followed by Bonferroni adjustments were performed to determine if there was a significant dose response for (i) the number of IL-8–positive cells in both cell lines, (ii) the number of thioredoxin-positive cells in both cell lines, (iii) the volume of IL-8 produced per BEAS-2B S.6 cell volume, and (iv) the volume of thioredoxin produced per BEAS-2B S.6 cell volume over the hydrogen peroxide concentrations (Systat 10.0; SPSS Inc., Chicago, IL). Nonparametric Kruskal-Wallis one-way ANOVA tests were performed to determine if there were significant differences between the uninjured and injured/IL-8–positive or thioredoxin-positive cells (e.g., Ethidium Homodimer-1–positive/IL-8–positive cells versus Ethidium Homodimer-1–negative/IL-8–positive cells) in both cell lines.

To determine if neutrophils colocalize with thioredoxin-positive cells, we analyzed the percentage of neutrophils binding with thioredoxin-positive compared with -negative cells above nonspecific binding conditions. The numbers of neutrophils touching or within a one-half epithelial cell diameter were combined and a Mann Whitney U test was performed. Results were considered significant when P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased Cell Injury and Oxidant Stress with Increasing H2O2 Dose
As expected, we saw increasing cellular and mitochondrial injury and oxidant stress with increasing H2O2 dose in the BEAS-2B S.6. Cellular injury was defined as Ethidium Homodimer-1–positive cells. Ethidium Homodimer-1 is cell impermeable until there is a defect in the cell membrane. Then the dye readily enters the cell, binds to nucleic acids, and fluoresces. Mitochondrial injury was defined as cells that were completely negative with MitoTracker dye. MitoTracker dye readily diffuses across cell membranes, where it accumulates and fluoresces in functional mitochondria. To investigate the response to oxidant stress, cells were loaded with the oxidant sensitive dye, 5-(and 6)-chloromethyl-2',7'-dicholorodihydrofluorescein diacetate before H2O2 exposure. This dye diffuses into cells, where subsequent oxidation yields a fluorescent adduct that is trapped in the cell. The dye detects a broad range of oxidizing reactions that may be increased during intracellular oxidant stress (20). Figure 1 shows significantly more BEAS-2B S.6 cells displayed cellular and mitochondrial injury with the 600 µM H2O2 dose compared with lower doses (P < 0.01 for 0, 200, and 400 versus 600 µM H2O2 for Ethidium homodimer; P < 0.01 for 0 and 200 versus 600 µM H2O2 for MitoTracker). As expected, more cells also displayed oxidant stress with increasing H2O2 concentration. Monolayers at 0 µM and 200 µM H202 sustained significantly less oxidant stress compared with higher H2O2 concentrations (0 versus 200, 400, and 600 µM; P < 0.01; 200 versus 400 and 600 µM; P < 0.01).



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  		Figure 1. Dose response of cellular oxidant stress and injury over H2O2 concentrations. Significantly more cells displayed cellular and mitochondrial injury with the 600 µM H2O2 dose compared with lower doses (0, 200, and 400 versus 600 µM H2O2, P < 0.01 for Ethidium homodimer; 0 and 200 versus 600 µM H2O2, P < 0.01 for MitoTracker cells). Monolayers at 0 µM and 200 µM H2O2 sustained significantly less oxidant stress compared with higher H2O2 concentrations (0 versus 200, 400, and 600 µM, P < 0.01; and 200 versus 400 and 600 µM, P < 0.01). Significance denoted by asterisks. Squares, Ethidium Homodimer; triangles, oxidant stress; X, MitoTracker-negative.

 
IL-8 and Thioredoxin Production Differs Depending on the Oxidant Injury Status of the Cell
To investigate how much oxidant stress or injury an airway epithelial cell can sustain and produce IL-8 or thioredoxin, cells were stained with several markers representing a range of injury. Very few injured BEAS-2B S.6 cells expressed the activated form of caspase-3 (data not shown).

Figure 2 is a summary of the staining observed with each marker of injury and IL-8 or thioredoxin. In both the TBE cells and BEAS-2B S.6, we found an inverse relationship between IL-8–positive cells and injured and stressed cells using all three markers of cell injury. That is, uninjured cells were significantly more likely to produce IL-8 compared with cells that were injured or oxidant-stressed (TBE cells: IL-8–positive/Ethidium Homodimer-1–positive versus IL-8–positive/Ethidium Homodimer-1–negative: P < 0.01 for 0, 200, and 400 and P < 0.02 for 600 µM H2O2; IL-8–positive/MitoTracker-positive versus IL-8–positive/MitoTracker-negative: P < 0.01 for 0, 200, and 400; IL-8–positive/oxidant stress–positive versus IL-8–positive/oxidant stress–negative: P < 0.01 for 0 and P = 0.041 for 200 µM H2O2. BEAS-2B S.6: IL-8–positive/Ethidium Homodimer-1–positive versus IL-8–positive/Ethidium Homodimer-1–negative: P < 0.01 for 0, 200, 400, and 600 µM H2O2; IL-8–positive/ MitoTracker-positive versus IL-8–positive/MitoTracker-negative: P < 0.01 for 0, 200, 400, and 600 µM H2O2; IL-8–positive/oxidant stress–positive versus IL-8–positive/oxidant stress–negative: P < 0.01 for 200, 400, and 600 µM H2O2).



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  		Figure 2. Summary of chemokine staining for each marker of oxidant stress or injury in the TBE cells (tracheobronchial epithelial cells) and BEAS cells (BEAS-2B S.6). (2-1) An oxidant stress–positive cell (green) is IL-8–negative, whereas a neighboring oxidant stress–negative cell is IL-8–positive (red). (2-2) The cell with functional mitochondria (red) is also IL-8–positive (green). The neighboring cell has no functional mitochondria and is IL-8–negative. (2-3) The injured, Ethidium Homodimer–1 positive, cell (red nucleus) is IL-8–negative. The neighboring cell is uninjured and IL-8–positive (green). (2-4A) Two cells are oxidant stress–positive (green) after exposure to 200 µM H2O2, whereas a neighboring cell is not oxidant-stressed (arrow). (2-4B) The same cells imaged the next day. The oxidant-stressed cells are IL-8–negative, whereas the unstressed cell is IL-8–positive (red staining, arrow). (2-5) The cell with functional mitochondria (red) is also IL-8–positive (green). The neighboring cell has no functional mitochondria and is IL-8–negative (arrow). (2-6) The injured Ethidium Homodimer-1–positive cell (red nucleus) is IL-8–negative. The neighboring cell is uninjured and IL-8–positive (green). (2-7A) Four oxidant stress–positive cells (green). (2-7B) The same field imaged the next day after staining for thioredoxin. One of the oxidant stress–positive cells is thioredoxin-positive (red), whereas another is thioredoxin-negative (arrowhead). The other two cells detached during staining. (2-8) The thioredoxin-positive cell (green) does not have functional mitochondria, whereas a neighboring cell with functional mitochondria (red) is thioredoxin-negative. (2-9) The injured, Ethidium Homodimer-1–positive, cell (red nucleus) is thioredoxin-negative. The neighboring cell is uninjured and thioredoxin-positive (green). (2-10A) Three oxidant stress–positive cells (green). (2-10B) The same field imaged the next day after staining for thioredoxin. One of the oxidant stress–positive cells is thioredoxin-positive (red), whereas the others are thioredoxin-negative (arrowheads point to the same cell). (2-11) The thioredoxin-positive cells (green) do not have functional mitochondria, whereas neighboring cells with functional mitochondria (red) are thioredoxin-negative. (2-12) The injured, Ethidium Homodimer-1–positive, cells (red nuclei) are thioredoxin-negative. The neighboring cells are uninjured and thioredoxin-positive (green).

 
In contrast, thioredoxin was more likely to be produced by airway epithelial cells that had sustained mitochondrial injury (TBE cells: thioredoxin-positive/MitoTracker-positive versus thioredoxin-positive/MitoTracker-negative: P < 0.01 for 200, 400, and 600 µM H2O2. BEAS-2B S.6: thioredoxin-positive/MitoTracker-positive versus thioredoxin-positive/MitoTracker-negative: P < 0.01 for 0, 200, 400, and 600 µM H2O2).

Cells that sustained cellular injury (Ethidium Homodimer-1–positive) did not produce thioredoxin (TBE cells and BEAS-2B S6: thioredoxin-positive/Ethidium Homodimer-1–positive versus thioredoxin-positive/Ethidium Homodimer-1–negative: P < 0.01 for 0, 200, 400, and 600 µM H2O2). These results imply that a cell can sustain only a certain degree of injury and still produce thioredoxin. If the injury is too severe, such as necrosis, the cell is unable to produce IL-8 or thioredoxin.

Our results with thioredoxin-positive cells and oxidant stress were equivocal. In the TBE cells, there was no significant difference in the oxidant stress status of the thioredoxin-positive cells. In the BEAS-2B S.6 cell line thioredoxin-positive cells were significantly more likely to be oxidant stress–negative than –positive only at 400 µM H2O2 (thioredoxin-positive/oxidant stress–positive versus thioredoxin-positive/oxidant stress–negative: P < 0.01). However, in both cell lines, oxidant-stressed cells were significantly more likely to produce thioredoxin than IL-8 after exposure to H2O2 (TBE cells: IL-8–positive/oxidant stress–positive versus thioredoxin-positive/oxidant stress–positive P < 0.01; BEAS-2B S.6: IL-8–positive/oxidant stress–positive versus thioredoxin-positive/oxidant stress–positive P < 0.01 for 400 and 600 µM H2O2). Taken together, these results indicate that cells need to sustain a certain degree of injury to upregulate thioredoxin. When exposed to a lesser degree of oxidant stress, a large number of cells will not produce thioredoxin. Alternatively, the dye that we used as an indicator of oxidant stress may not have been a sensitive indicator of when a stressed cell will upregulate thioredoxin.

Tables 1 and 2 summarize the differential cell counting in the TBE cells after H2O2 exposure. Table 1 summarizes the percentage of cells stained with each marker of injury or stress and thioredoxin over all H2O2 concentrations (mean percentage ± SE) and Table 2 summarizes the percentage of cells stained with the same markers and IL-8 (mean percentage ± SE). As with the data expressed in mm2, nonparametric Kruskal-Wallis one-way ANOVA tests were performed on this data with no differences in the outcome of significance.


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  		TABLE 1. Summary of differential cell counting in TBE cells after H2O2 exposure and staining for each marker of cell injury and thioredoxin (mean percentage ± SE)

 

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  		TABLE 2. Summary of differential cell counting in TBE cells after H2O2 exposure and staining for each marker of cell injury and IL-8 (mean percentage ± SE)

 
A Significant Inverse Relationship between the Number of IL-8 and Thioredoxin-Positive Cells with Increasing H2O2 Dose
Figures 3A and 3B demonstrate that whereas the number of IL-8–positive cells decreased with increasing H2O2 dose, the number of thioredoxin-positive cells increased in both cell lines. Figure 3A demonstrates that in both the TBE cells and BEAS-2B S.6, there were significantly more IL-8–positive cells at 0 µM H2O2 compared with higher H2O2 doses (TBE cells: 0 versus 400 and 600 µM, P < 0.01; BEAS-2B S.6: 0 versus 200 µM, P = 0.02; 0 versus 400 µM, P = 0.03; 0 versus 600 µM, P < 0.01). Figure 3B shows that TBE cells produced significantly more thioredoxin only at 400 µM H2O2 compared with 0 µM H2O2 (P = 0.039) whereas the BEAS-2B S6 cells produced significantly more thioredoxin at 600 µM H202 compared with the lower doses (P < 0.01). In the BEAS-2B S.6, both a Spearman partial correlation and Kendall {tau}b partial correlation showed an overall significance for a negative relationship between the number of IL-8–positive and thioredoxin-positive cells over all the H2O2 concentrations (P < 0.01). These results demonstrate that there is a significant inverse relationship between IL-8–positive cells and thioredoxin-positive cells relative to oxidant injury. That is, airway epithelial cells are significantly more likely to retain IL-8 with no or minimal exposure to H2O2 but more likely to produce thioredoxin when they are exposed to higher concentrations of H2O2.




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  		Figure 3. Number of IL-8 and thioredoxin-positive cells per mm2 surface area in both cell types over all H2O2 concentrations. (A) The number of IL-8–positive cells decreased with increasing H2O2 dose (TBE cells: 0 versus 400 µM and 0 versus 600 µM, P < 0.01; BEAS-2B S.6: 0 versus 200 µM P = 0.02, 0 versus 400 µM P = 0.03, 0 versus 600 µM P < 0.01). Squares, IL-8 TBE; triangles, IL-8 BEAS. (B) The number of thioredoxin-positive cells increased with increasing H2O2 dose (TBE cells: 0 versus 400 µM, P = 0.039; BEAS-2B S.6: 0 versus 200, 400, and 600 µM P < 0.01). Squares, THX TBE; triangles, THX BEAS. Significance denoted by asterisks.

 
Individual BEAS-2B S.6 Cells Produce Less IL-8 and More Thioredoxin with Increasing H2O2 Dose
To investigate if there was a dose response in the amount of cytokine an individual cell produces in response to an oxidant insult, the volume of IL-8 or thioredoxin per BEAS-2B S.6 cell was estimated at each dose of H2O2 (Vcytokine/Vcell). Monensin (GolgiStop) was used in all the cell culture media to block intracellular protein transport and result in the accumulation of chemokines in the Golgi complex. Thus, the net accumulation of IL-8 and thioredoxin over the 8-h period was estimated. Figure 4A demonstrates that the volume of IL-8 per cell volume decreased with increasing H2O2 concentration. Cells exposed to 0 µM and 200 µM produced significantly more IL-8 than cells at 400 µM and 600 µM H2O2 (0 versus 400 and 600 µM, P < 0.01; 200 versus 400 µM, P = 0.01; 200 versus 600 µM, P < 0.01). Figure 4B demonstrates the opposite effect for thioredoxin. That is, the volume of thioredoxin per cell volume increased with increasing H2O2 concentration. Cells exposed to 400 µM and 600 µM produced significantly more thioredoxin than cells at 0 µM and 200 µM H2O2 (600 versus 0, 200, and 400 µM, P < 0.01; and 400 versus 0 and 200 µM, P < 0.01). Our data show that with increasing H2O2 concentration, cells produce less IL-8 and more thioredoxin.




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  		Figure 4. Volume of IL-8 and thioredoxin staining per cell volume at each H2O2 concentration in BEAS-2B S.6 cells. (A) Cells exposed to 0 µM and 200 µM produced significantly more IL-8 than cells at 400 µM and 600 µM H2O2 (0 versus 400 and 600 µM, P < 0.01; 200 versus 400 µM, P = 0.01; 200 versus 600 µM, P < 0.01). (B) Cells exposed to 400 µM and 600 µM produced significantly more thioredoxin than cells at 0 µM and 200 µM H2O2 (600 versus 0, 200, and 400 µM, P < 0.01; 400 versus 0 and 200 µM, P < 0.01). Significance denoted by asterisks.

 
No Observable Injury to the Golgi Apparatus with H2O2 Incubation
To assess the integrity of the Golgi apparatus after hydrogen peroxide incubation, we performed two control experiments in the BEAS-2B S.6 cells. First, we used a Golgi specific dye to visualize the Golgi after exposure to 600 µM H2O2 compared with 0 µM H2O2 and found no difference in staining characteristics between these groups (data not shown). Second, after exposing cells to either 0 or 600 µM H2O2, cells were strongly stimulated for IL-8 production with TNF-{alpha} and IL-1ß in the presence of monensin for 7 h similar to the time course of these experiments. There was no significant difference in the number of IL-8–positive cells between the hydrogen peroxide doses (data not shown). These data indicate that the Golgi apparatus remains intact with negligible leakage of IL-8 even after exposure to 600 µM H2O2.

Human Neutrophils Colocalized with Thioredoxin-Positive BEAS-2B S.6 Cells
We investigated whether neutrophils migrate to and associate with thioredoxin-positive cells. We accounted for nonspecific neutrophil binding to epithelial monolayers by counting the number of neutrophils associated with uninjured, predominantly thioredoxin-negative BEAS-2B.S.6. Figure 5 shows that neutrophils colocalized significantly more often with thioredoxin-positive cells compared with thioredoxin-negative cells expressed as a percentage change from baseline nonspecific binding (P < 0.01). These results indicate that neutrophils can specifically associate with BEAS-2B S.6 cells producing thioredoxin.



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  		Figure 5. Neutrophil colocalization with thioredoxin-positive and thioredoxin-negative BEAS-2B S.6 cells expressed as a percentage change above nonspecific binding. Neutrophils were significantly more likely to colocalize with thioredoxin-positive cells compared with thioredoxin-negative cells (P < 0.01). Significance denoted by an asterisk.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we investigated the phenotype of the airway epithelial cell that produces IL-8 and thioredoxin at the individual cell level after an oxidant injury in two different cell lines. Specifically, we investigated the degree of injury or stress that an airway epithelial cell could sustain and produce either IL-8 or thioredoxin. To our knowledge, this is the first paper to correlate the state of cell injury or stress with chemokine production at the individual airway epithelial cell level. For this study, we chose to use hydrogen peroxide as the oxidant and a dose range (0–600 µM) that resulted in a good distribution of injured and noninjured cells. Additionally, we used monensin, an intracellular protein transport inhibitor, in all the cell culture media to prevent secretion of chemokines. Thus, the chemokines accumulated within individual cells from the time of injury through the postexposure period. This permitted good visualization of positive cells and allowed us to quantify the number of positive cells and the volume of chemokines produced during this period.

We found similar results in both primary human airway epithelial cells and a transformed cell line, BEAS-2B S.6. Neither oxidant-injured nor oxidant-stressed airway epithelial cells produced significant amounts of IL-8. Similarly, cells that sustained a large degree of cellular injury (Ethidium Homodimer-1–positive) did not produce thioredoxin. These results indicate that there is a limit to the degree of injury a cell can sustain and produce either IL-8 or thioredoxin. Necrotic cells did not produce IL-8 or thioredoxin. However, in contrast to IL-8, airway epithelial cells with mitochondrial injury (MitoTracker-negative) did produce and upregulate thioredoxin. Likewise, oxidant-stressed airway epithelial cells were more likely to produce thioredoxin than IL-8. These results indicate that airway epithelial cells produce different chemokines depending on their degree of oxidant injury or stress that may play distinct roles in navigating neutrophils to a site of oxidant injury.

To reach a site of injury, neutrophils must travel out of the vasculature, across a connective tissue space, and in some cases, across an epithelial barrier. Some investigators have begun to define a multistep paradigm with different chemokines creating distinct gradients to guide neutrophils to the site of injury. Foxman and colleagues found that neutrophils were able to effectively chemotax to a secondary distant signal after migrating up a primary gradient and reaching a saturated concentration. They proposed a multistep model of chemoattractant-directed migration requiring leukocytes to respond to multiple serial signals with distinct microenvironmental localization (21). In fact, using a model of intestinal epithelium colonized by Salmonella typhimurium, McCormick and colleagues have proposed a paradigm in which epithelial cells release chemokines, including IL-8, basolaterally to recruit neutrophils across the subepithelial matrix. Recently, they found S. typhimurium attachment to apical epithelial membranes induced apical release of pathogen-elicited epithelial chemoattractant (PEEC), which may direct neutrophil movement across the epithelium (22, 23).

Similar to intestinal epithelium, there is evidence that multiple chemoattractant gradients are necessary to guide neutrophils up to and then across an airway epithelial barrier. Like intestinal epithelium, basolateral secretion of IL-8 has been shown in other types of epithelium, including retinal and type II epithelial cells (24, 25). Numerous studies have shown the importance of IL-8 in recruiting neutrophils to sites of injury, although it may not be an important mediator in drawing neutrophils across an epithelial barrier. Miller and colleagues found that migration of neutrophils across a monolayer of airway epithelial cells was dependent on both thioredoxin and pertussis toxin–sensitive mediators, but was independent of IL-8 (6). Their study demonstrated that thioredoxin, not IL-8, is an important mediator for neutrophil transepithelial migration.

Once neutrophils arrive at a site of epithelial injury, evidence suggests that they are important in removing injured airway epithelial cells and hastening epithelial repair. In a series of experiments using in vitro type II alveolar cells, rats, and rhesus monkeys, Hyde and colleagues found that neutrophils enhance the repair of ozone-injured airway epithelium (2, 26, 27). In one study, rhesus monkeys were exposed to 0.8 ppm ozone and administered a function-blocking monoclonal antibody against CD18 to prevent neutrophil emigration. They found a significant number of necrotic airway epithelial cells when neutrophil influx was blocked; however, when neutrophils were recruited back into the contralateral lung with instillation of C5a, there were significantly fewer necrotic cells. They concluded that neutrophils contribute to the repair of oxidant-injured airway epithelium by removing injured epithelial cells. Our findings that neutrophils colocalize with thioredoxin-positive airway epithelial cells, which are likely to be oxidant-injured or -stressed, suggests that thioredoxin production, at least in part, is important in this neutrophil-dependent removal.

We propose that IL-8 and thioredoxin may be creating two distinct gradients guiding neutrophils to a site of oxidant injury. IL-8, produced by uninjured neighboring cells, may create a primary gradient to guide neutrophils out of the vasculature and across the connective tissue, whereas thioredoxin, produced in part by oxidant-injured and -stressed airway epithelial cells, may create a secondary gradient guiding neutrophils across the airway epithelium and target oxidant-injured airway epithelial cells for neutrophil-dependent removal.

Thioredoxin production by oxidant-injured and -stressed airway epithelial cells may induce healthy neighboring cells to upregulate IL-8. Several investigators have established that thioredoxin acts to regulate the expression of other cytokines. At low concentrations, thioredoxin strongly stimulates mRNA and protein production of IL-1, IL-2, TNF, and IL-8 in several different cell types. Thioredoxin induced the human monocytic cell line, Mono Mac6, to increase IL-8 mRNA after phorbolester stimulation (13). Additionally, IL-8 promoter activity was enhanced in BEAS-2B airway epithelial cells after treatment with thioredoxin (14). Both of these investigators have shown that thioredoxin activates NF-{kappa}B and AP-1 transcription factors that are essential in transcriptional control of cytokine genes, including IL-8 (28).

Many studies showing that oxidants, such as H2O2, increase IL-8 production, may seem contradictory to our findings that IL-8 is not produced by oxidant-stressed airway epithelial cells. DeForge and colleagues found that oxidant stress was an important regulator for IL-8 gene expression (29). They found that exposure of Hep-G2 cells, A549 pulmonary type II epithelial cells, and human skin fibroblasts to H2O2 stimulated IL-8 production. Part of the discrepancy in results may be due to the different cell types used in these studies. Furthermore, prior studies investigated populations of cells and did not study the oxidant stress status of the individual IL-8–producing cell. Hence, although there maybe an overall increase in IL-8 production by tissues or populations of cells induced by oxidant stress, the individual oxidant-stressed cells are not primarily responsible for the increase.

In conclusion, our findings support the hypothesis that neither oxidant-injured nor oxidant-stressed airway epithelial cells produce IL-8, but they upregulate thioredoxin. We believe that these data are important in a multistep navigation of neutrophils to a site of oxidant injury.


   Acknowledgments
 
The authors thank Neil Willits for statistical advice. They also thank Erica Gurley and Mara Schnayer for their laboratory assistance. Yu Hua Zhao expertly prepared all the human tracheobronchial epithelial cells for use in these experiments. This study was supported by NIH Grant ES00628, ES09701, and HL35635.

Received in original form November 25, 2002

Received in final form September 16, 2003


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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