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* From the Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, CO.
Correspondence to: Milene Saavedra, MD, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Ave, Box C272, Denver, CO 80262; e-mail: Saavedra{at}UCHSC.edu
A unique host pathogen relationship exists between Pseudomonas aeruginosa and human airway epithelium (HAE) in patients with cystic fibrosis. Chronic colonization of P aeruginosa in cystic fibrosis airways is a major source of morbidity and mortality in these patients. Iron (Fe) availability regulates the virulence of P aeruginosa, but the effect of altered Fe availability on the host-pathogen relationship is not known. P aeruginosa is an obligate respirer, utilizing oxygen or nitrate. It cannot perform its respiratory functions without Fe. This need for Fe also places the bacterium at a high potential for Fe-catalyzed oxidative stress. P aeruginosa specifically requires reduced Fe2+, a soluble form of Fe. In the microenvironments of lungs and blood, high oxygen concentrations lead to formation of the oxidized insoluble form of Fe, specifically Fe3+. Fe is further made inaccessible by binding to transferrin and lactoferrin. P aeruginosa requires Fe2+ concentrations of 10-6 M to survive, whereas it encounters environmental concentrations of 10-18 M. The pursuit of Fe is mediated through various mechanisms including: (1) production of proteases to degrade host Fe binding compounds; (2) release of Fe binding compounds, siderophores, which bind the free Fe3+ released from host lactoferrin and transferrin following exposure to proteases; (3) reduction of insoluble Fe3+ to soluble Fe2+ by membrane reductases; and (4) production of compounds such as exotoxins that are toxic to eukaryotic cells, eliminating competition for usable Fe.1
In this study, we tested the hypothesis that P aeruginosa grown under low-Fe conditions would induce more injury to human airway epithelial cells than P aeruginosa grown under high-Fe conditions. We present evidence that high-Fe P aeruginosa infection of airway epithelial cells results in increases in both inflammatory and cell protective genes on a transcriptional level. On a translational level, low-Fe-infected PAO1 cells demonstrate a more inflammatory program than high-Fe P aeruginosa. However, when we evaluated cell death, higher rates of cell death were evident with the high-Fe-infected P aeruginosa, as well as higher rates of apoptosis.
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Materials and Methods |
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In order to confirm that the PAO1 remained in either low-FE and high-Fe states in tissue culture media, a lacZ-exotoxin A fusion was generated (exotoxin A is produced only under low-Fe conditions). Next, ß-galactosidase assays done for PAO1 exotoxin A revealed low-Fe PAO1 maintained exotoxin A production in tissue culture media and that high-Fe PAO1 had no exotoxin A production in tissue culture media.
Transcriptional and Translational Responses
Complementary DNA was prepared from RNA and quantitative reverse
transcriptase-polymerase chain reaction was performed using SYBR green
(Perkin-Elmer 5700; PE Biosystems; Foster City, CA). Enzyme-linked
immunosorbent assay was performed on both low-FE-infected and
high-FE-infected cell supernatants for both tumor necrosis factor and
monocyte chemotactic protein.
Apoptosis Detection
The TACS Annexin V-FITC apoptosis detection system (R&D
Systems; Minneapolis, MN) uses annexin V-fluorescein isothiocyanate
(FITC) conjugates for flow cytometry detection of apoptotic
cells. Cells are harvested, washed, and then incubated with annexin
V-FITC and propidium iodide. They are subsequently analyzed by flow
cytometry. The combination of annexin V-FITC and propidium iodide
allows for differentiation between early apoptotic cells (annexin V
positive, propidium iodide negative), late apoptotic and/or necrotic
cells (annexin V and propidium iodide positive), and viable cells
(unstained).
Apoptosis detection on cells was completed using a modified deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (DeadEnd Colorimetric Apoptosis Detection System; Promega Corp; Madison, WI). The nicked 3'OH DNA ends incorporate biotinylated nucleotide using terminal deoxynucleotidyl transferase. After addition of horseradish peroxidase-labeled streptavidin, these nucleotides are then detected with hydrogen peroxide and the stable chromogen, diaminobenzidine. The apoptotic cells stain dark brown (Fig 1 ).
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Results and Discussions |
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B was
induced twofold in low-Fe-infected vs high-Fe-infected cells. However,
cell protective genes (specifically, the protease inhibitors secretory
leukocyte protease inhibitor, elastase specific protease inhibitors,
and monocyte neutrophil elastase inhibitor) are preferentially
upregulated in the high-Fe PAO1, from 2.5-fold to sevenfold.
Because PAO1 invades via disruption of tight junctions, we also
evaluated for changes in connexin and zona occludens regulation with no
evidence of change on a message level. We evaluated this
transcriptional difference in inflammation on a protein basis with the
use of enzyme-linked immunosorbent assays, which demonstrated
significant increases in both tumor necrosis factor-
and monocyte
chemotactic protein-1 in the low-Fe PAO1 infected airway cells.
In our model, we have demonstrated that epithelial cells are destroyed, depending on the PAO1 multiple of infectivity, at anywhere from 6 to 12 h. We subsequently evaluated rates of epithelial cell death depending on low-Fe vs high-Fe PAO1 infection.
Both annexin and propidium iodide staining demonstrated increased cell death in cells infected with high-Fe PAO1. Serial dilutions demonstrated no significant differences in bacterial cell counts after 1 h. Since the only difference in the faster dying cultures was the presence of Fe, the role of oxidative stress was investigated. The presence of free Fe (Fe 2+/Fe 3+) catalyzes the Fenton reaction in which hydrogen peroxide and oxygen free radicals form hydroxyl free radicals, which cause oxidative damage to DNA, protein, and lipids. We pretreated our cells with tetrakis (4-benzoic acid) porphyrin (TBAP), a cell permeant superoxide scavenger that mimics superoxide dismutase activity. This eliminates oxygen radicals, preventing the formation of free hydroxyl radicals. Flow cytometry analysis for propidium iodide-positive cells revealed that addition of TBAP made no difference in cell survival with mortality rates of 40 to 60% and a 5% mortality in TBAP control. These rates are comparable to non-TBAP-treated cells that have been infected. High-Fe-infected and low-Fe-infected cells were then evaluated by TUNEL assay with marked differences in percentage apoptotic cells. High-Fe cells demonstrated 50% apoptosis vs 7% in low-Fe PAO1 infected cells.
The implications that arise from this data are twofold. First, low-Fe-infected HAE manifest a more inflammatory program on a translational level. Not only do high-Fe-infected HAE exhibit slightly higher expression of inflammatory cytokines, they demonstrate markedly greater expression of protease inhibitors than do the low-Fe-infected HAE. Second, high-Fe-infected HAE die at accelerated rates compared to Fe-deficient, P aeruginosa-infected HAE. Since rates of apoptosis are dramatically higher in these cells, one possibility is that apoptosis is an adaptive response to PAO1 infection among HAE. Further experiments are necessary to elucidate how Fe affects the apoptotic response.
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Footnotes |
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This article has been cited by other articles:
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  D.W. Reid, Q.T. Lam, H. Schneider, and E.H. Walters Airway iron and iron-regulatory cytokines in cystic fibrosis Eur. Respir. J., August 1, 2004; 24(2): 286 - 291. [Abstract] [Full Text] [PDF]
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