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. 2004 Nov;45(11):4066-74.
doi: 10.1167/iovs.04-0627.

Hypoxia increases corneal cell expression of CFTR leading to increased Pseudomonas aeruginosa binding, internalization, and initiation of inflammation

Tanweer Zaidi  1 , Mary Mowrey-McKee, Gerald B Pier
Affiliations

Hypoxia increases corneal cell expression of CFTR leading to increased Pseudomonas aeruginosa binding, internalization, and initiation of inflammation

Tanweer Zaidi et al. Invest Ophthalmol Vis Sci. 2004 Nov.

Abstract

Purpose: To investigate the effect of hypoxia-induced molecular responses of corneal epithelial cells on the surface of rabbit and human corneas and corneal cells in culture on interactions with Pseudomonas aeruginosa that may underlie increased susceptibility to keratitis.

Methods: Organ cultures of rabbit and human corneal tissue, primary rabbit and human corneal cells, and transformed human corneal cells from a patient with cystic fibrosis and the same cell line corrected for expression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), the cellular receptor for P. aeruginosa, were exposed to hypoxic conditions for 24 to 72 hours. Changes in binding and internalization of P. aeruginosa were measured using cellular association and gentamicin-exclusion assays, and expression of CFTR and activation of NF-kappaB in response to hypoxia were determined by confocal laser microscopy and quantitative measurements of NF-kappaB activation.

Results: Hypoxia induced in a time- and oxygen-concentration-dependent manner increased association and internalization of clinical isolates of P. aeruginosa in all cells tested. Hypoxia increased CFTR expression and NF-kappaB nuclear translocation in rabbit and human cells with wild-type CFTR. Corneal cells lacking CFTR had reduced NF-kappaB activation in response to hypoxia. Hypoxia did not affect the increase in corneal cell CFTR levels or NF-kappaB activation after P. aeruginosa infection.

Conclusions: Hypoxic conditions on the cornea exacerbate the binding and internalization of P. aeruginosa due to increased levels of CFTR expression and also induce basal NF-kappaB activation. Both of these responses probably exacerbate the effects of P. aeruginosa infection by allowing lower infectious doses of bacteria to induce disease and promote destructive inflammatory responses.

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Figures

Figure 1
Figure 1
Effect of hypoxia on association and internalization of P. aeruginosa by the corneal epithelia on rabbit eyes in organ culture. (A–C) Levels of P. aeruginosa strain 6294 associated with corneal cells on intact rabbit corneas incubated in the indicated level of oxygen for the indicated time period. (D, E) Levels of cytotoxic P. aeruginosa strain 6206 associated (D) with or internalized (E) by corneal cells on an intact rabbit cornea incubated for the indicated times in 20% or 15% oxygen. (F) Associated and internalized levels of P. aeruginosa PAO1 by corneal cells on an intact rabbit cornea incubated for 48 hours in 20% or 15% oxygen. Bars, means of four to six samples; error bars, SEM. *P < 0.05, t-test when comparing cells kept in 20% oxygen with cells kept in lower concentrations of oxygen.
Figure 2
Figure 2
Effect of hypoxia on the corneal surface morphology rabbit eyes. Rabbit eyes were incubated for the indicated time in either 20% or 15% oxygen. Shown are areas representative of a significant portion of the corneal surface. Incubation in 15% oxygen showed cellular disruption, loosening of junctions and exposure of basolateral surfaces along with cellular desquamation. Magnification, ×400.
Figure 3
Figure 3
Reversibility of the effect of hypoxia on the association of P. aeruginosa strain 6294 with rabbit corneas. P. aeruginosa association and internalization into the corneal cells was determined after continuous incubation in 20% or 15% oxygen, or 15% oxygen for 24 hours then 20% oxygen for either an additional 24 or 48 hours, giving a total incubation time of 48 and 72 hours, respectively, for these corneas. Bars, means of four to six samples; error bars, SEM. *P < 0.05, t-test for two-group comparisons as well as ANOVA for three-group comparisons, along with P <.05 for the Fisher PLSD for pair-wise comparisons. Comparisons are for results from cells indicated by asterisks with results from cells in the same grouping incubated continuously in 20% oxygen.
Figure 4
Figure 4
Effect of hypoxia on association and internalization by cultured cells of P. aeruginosa strain 6294. Associated (A on x-axes) and internalized (I on x-axes) P. aeruginosa 6294 in rabbit (A) or human (B) primary corneal epithelial cells in cultures incubated for 48 hours in either 20% or 15% oxygen. (C) Association and internalization of P. aeruginosa 6294 into transformed human corneal cells expressing either wild-type or ΔF508 CFTR incubated in either 20% or 15% oxygen. (D) Inhibition of internalization by wild-type or ΔF508 CFTR transformed human corneal cells in culture in 20% oxygen by peptide 108–117 of CFTR but not by a scrambled version of this same peptide. (D) The probability is for both overall ANOVA and Fisher’s PLSD post hoc comparison of the effects of peptide 108–117 with both no peptide and scrambled peptide. (E) NF-κB p65 subunit nuclear translocation 15 minutes after exposure to P. aeruginosa 6294 of transformed human corneal cells expressing either wild-type or ΔF508 CFTR. NF-κB p65 in the nucleus is shown in magenta, representing the colocalization of NF-κB p65, visualized by a red-dye–conjugated reagent, and the blue nucleus, visualized by staining with DAPI. Magnification, ×800.
Figure 5
Figure 5
CFTR expression in rabbit and human corneas after 24 or 48 hours of incubation in atmospheric or 15% oxygen. Expression of CFTR in rabbit and human corneas, primary corneal cells, or transformed corneal cells expressing wild-type (WT) CFTR was increased after culture for 48 hours in 15% oxygen compared with culture in 20% oxygen. In transformed human corneal cells expressing ΔF508 CFTR, a small but perceptible increase in the amount of the mutant protein in cells exposed to hypoxic conditions was noted.
Figure 6
Figure 6
Effect of hypoxia on translocation of NF-κB in rabbit or human corneal epithelium or primary corneal cell cultures. After the indicated time of incubation in 20% or 15% oxygen, the presence of active NF-κB in the nucleus of both the corneal tissue and the cells was visualized by immunohistochemical staining. NF-κB p65 subunit in the nucleus is stained magenta, representing the colocalization of NF-κB p65, visualized by a red-dye–conjugated reagent, and the blue nucleus, visualized by staining with DAPI. Magnification: corneal epithelium, ×100; primary epithelial cells, ×400.
Figure 7
Figure 7
Effect of oxygen concentration on the amount of activated NF-κB p65 subunit present in the nuclei of corneal cells. Transformed human corneal cells, expressing either wild-type or ΔF508 CFTR, or primary human corneal cells were incubated for 48 hours in 20% or 15% oxygen without infection (A) or after infection (B) with P. aeruginosa. Bars, means of triplicate samples; error bars, SEM. Probabilities were derived by comparison of amount of activated NF-κB in cells exposed to 15% oxygen with that in cells exposed to 20% oxygen, using the Fisher PLSD determination for pair-wise comparisons after ANOVA, which generated, overall, P < 0.05.
Figure 8
Figure 8
Effect of hypoxia on expression of CFTR and NF-κB nuclear translocation after infection with P. aeruginosa. Comparable CFTR expression and NF-κB p65 subunit nuclear translocation were observed in rabbit or human corneal tissues, or primary rabbit or human corneal cells, incubated in either atmospheric (20%) or 15% oxygen for 48 hours followed by 3 hours of infection with P. aeruginosa. CFTR expression (green) and NF-κB p65 in the nucleus (magenta) were visualized by immunohistochemical staining. NF-κB p65 in the nucleus is stained magenta, representing the colocalization of NF-κB p65, visualized by a red-dye–conjugated reagent, and the blue nucleus, visualized by staining with DAPI. Magnification: corneas, ×100; corneal cells, ×400.
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