Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa

Abstract

Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model with P. aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains of P. aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.

Figures

FIG. 1

IL-6 mRNA expression in the corneas of mice during experimental infection with P. aeruginosa 6294, 6206, or Paer1 at various times. All of the micrographs are at the same magnification, except for the insets, which are at ×1,620. Not all sections are counterstained. Bar, 10 μm. (A) Cornea immediately after challenge with P. aeruginosa 6294, showing IL-6 mRNA expression. (B) Increased IL-6 mRNA expression in the corneal epithelium 1.5 h after challenge with P. aeruginosa 6294. (C) High levels of IL-6 mRNA expression in the cornea 4 h after challenge with P. aeruginosa 6294. (D) Significantly edematous cornea 24 h after infection with P. aeruginosa 6294. Large numbers of inflammatory cells have infiltrated the cornea, with destruction. IL-6 mRNA expression is significantly induced. The inset shows a higher magnification of IL-6 mRNA-positive cells in the stroma at the wound site. (E) Cornea immediately after challenge with strain 6206, showing IL-6 mRNA expression. (F) IL-6 mRNA expression in the corneal epithelium 1.5 h after challenge with strain 6206. (G) Higher levels of IL-6 mRNA expression in the cornea 4 h after challenge with strain 6206. (H) Cornea at 24 h after challenge with strain 6206. Large numbers of inflammatory cells have infiltrated the cornea. There is a loss of epithelial cells at the wound site. Inflammatory cells have also infiltrated the anterior chamber. The inset shows a higher magnification of IL-6 mRNA-positive cells in the stroma at the wound site. (I) Cornea immediately after challenge with Paer1, showing IL-6 mRNA expression. (J) mRNA expression 1.5 h after challenge with Paer1. (K) Induction of IL-6 mRNA expression at 4 h after challenge with Paer1. (L) At 24 h after challenge with Paer1, the cornea shows levels of IL-6 induction comparable to those elicited by PBS. The healed wound site can be seen. The inset shows a higher magnification of keratocytes staining positively for IL-6 mRNA. (M) Hematoxylin-eosin staining of the cornea 24 h after infection with 6294, showing sloughing off of the epithelium at the site of wounding (W) and infiltrating inflammatory cells. The inset shows a higher magnification of infiltrating cells, confirming that they are predominantly neutrophils (N) and that the IL-6 mRNA-positive cells in panel D are largely infiltrating inflammatory cells. (N) Hematoxylin-eosin staining of the cornea 24 h after infection with 6206, showing sloughing off of the epithelium at the site of wounding (W) and infiltrating inflammatory cells. The inset shows a higher magnification of infiltrating cells, confirming that they are predominantly neutrophils (N) and that the IL-6 mRNA-positive cells in panel H are largely infiltrating inflammatory cells. (O) Hematoxylin-eosin staining of the cornea 24 h after challenge with Paer1. The scratch site is fully reepithelialized. The inset shows a higher magnification of keratocytes (K) and occasional neutrophils associated with small focal infiltrates at the wound site. (P) Typical cornea hybridized with Dig-labelled control probe (sense), showing no positive staining in any part of the cornea. (Q) Cornea 24 h after wounding and topical application of PBS, showing levels of IL-6 mRNA induction comparable to those seen at 0 h for all strains.

FIG. 2

Changes in IL-6 mRNA expression in the corneas of mice after challenge with three strains of P. aeruginosa, 6294, 6206, and Paer1, over time. The amount of IL-6 mRNA was semiquantitated by image analysis and is expressed as image analysis units per section. An asterisk indicates that the value was significantly different from control and 0-h values. Data represent mean image analysis units for three eyes per time point, and error bars represent standard errors of the mean.

FIG. 3

Amounts of IL-6 protein detected in homogenates of whole eyes of mice infected with strain 6294, 6206, or Paer1. Note the increased presence of IL-6 24 h after infection with strains 6294 and 6206 only. See the legend to Fig. 2 for an explanation of asterisks. Data represent the mean for three eyes per time point, and error bars represent standard errors of the mean.