Andrew Wahlert

Functional Foxp3+ CD4+ CD25(Bright+) “Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4+ and CD8+ Effector T Cells during Ocular Herpes Infection

ABSTRACT We studied the phenotype and distribution of “naturally” occurring CD4+ CD25+ T regulatory cells (CD4+ CD25+ nTreg cells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (Teff) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45+ pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4+ CD25(Bright+) T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4+ CD25(Bright+) T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4+ CD25(Bright+) T cells, but not CD4+ CD25(low) T cells, efficiently suppressed HSV-specific CD4+ and CD8+ Teff cells. The CD4+ CD25(Bright+) T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating Teff cells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor β. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3+ CD4+ CD25(Bright+) T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3+ CD4+ CD25(Bright+) Treg cells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4+ CD25+ nTreg cells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.

Age-related changes in the meibomian gland.

The purpose of this study was to characterize the age-related changes of the mouse meibomian gland. Eyelids from adult C57Bl/6 mice at 2, 6, 12 and 24 months of age were stained with specific antibodies against peroxisome proliferator activated receptor gamma (PPARgamma) to identify differentiating meibocytes, Oil Red O (ORO) to identify lipid, Ki67 nuclear antigen to identify cycling cells, B-lymphocyte-induced maturation protein-1 (Blimp1) to identify potential stem cells and CD45 to identify immune cells. Meibomian glands from younger mice (2 and 6 months) showed cytoplasmic and perinuclear staining with anti-PPARgamma antibodies with abundant ORO staining of small, intracellular lipid droplets. Meibomian glands from older mice (12 and 24 months) showed only nuclear PPARgamma localization with less ORO staining and significantly reduced acinar tissue (p < 0.04). Acini of older mice also showed significantly reduced (p < 0.004) numbers of Ki67 stained nuclei. While Blimp1 appeared to diffusely stain the superficial ductal epithelium, isolated cells were occasionally stained within the meibomian gland duct and acini of older mice that also stained with CD45 antibodies, suggesting the presence of infiltrating plasmacytoid cells. These findings suggest that there is altered PPARgamma receptor signaling in older mice that may underlie changes in cell cycle entry/proliferation, lipid synthesis and gland atrophy during aging. These results are consistent with the hypothesis that mouse meibomian glands undergo age-related changes similar to those identified in humans and may be used as a model for age-related meibomian gland dysfunction.

Corneal Response to Femtosecond Laser Photodisruption in the Rabbit

In this report we evaluated the effect of femtosecond laser energy on the development of corneal haze and keratocyte activation in rabbits following intra-stromal photodisruption to create LASIK flaps using a modified commercial femtosecond surgical laser. Three groups of flap parameters were studied: 1.5 microJ/pulse with 10 microm spot separation and complete side cut (Group 1); 3.5 microJ/pulse with 14 microm spot separation and complete side cut (Group 2); 3.5 microJ/pulse with 14 microm spot separation and partial (50 microm) side cut (Group 3). All flaps were left attached without lifting to avoid epithelial contamination. Rabbits were then evaluated pre- and post-operatively by quantitative in vivo and ex vivo confocal microscopy. The achieved flap thickness 1 week after surgery averaged 88.9+/-12.8, 90.8+/-6.9 and 86.5+/-6.8 microm for Groups 1-3 respectively (p=NS). Interface thickness was significantly greater (p<0.05) in the higher energy groups averaging 40.0+/-11.2 and 37.7+/-5.7 microm for Groups 2-3 compared to 28.6+/-4.5 microm for Group 1. Corneal haze was barely detectible and not significantly different between groups, although haze was detected in the region of the side-cuts in Groups 1 and 2. No clinically significant changes in stromal or epithelial thickness were noted. Laser confocal microscopy showed the presence of small diameter cells within the flap interface that resided within disrupted regions of the corneal collagen lamellae. Keratocyte activation was only detected in regions of the 100% side cut and not over the flap interface. In conclusion, the results of this study indicate that photodisruption of the corneal stroma alone without flap elevation regardless of laser energy does not induce significant corneal haze in the rabbit. However, a thicker stromal interface was seen with the higher energy suggesting greater stromal damage.

Detection of Corneal Fibrosis by Imaging Second Harmonic–Generated Signals in Rabbit Corneas Treated with Mitomycin C after Excimer Laser Surface Ablation

PURPOSE Recent studies have shown that confocal imaging of second harmonic-generated (SHG) signals can detect corneal collagen organization. The purpose of this study was to assess whether SHG signals can detect differences in corneal fibrosis after excimer laser surface ablation (photorefractive keratectomy [PRK]). METHODS Rabbits received 9-D PRK in one eye followed by treatment with either mitomycin C (MMC) or vehicle. Corneal haze was measured by in vivo confocal microscopy before and 2, 4, 8, and 12 weeks after surgery. Animals were then killed and corneas were evaluated by visible and nonlinear confocal microscopy. RESULTS PRK induced significant haze in vehicle-treated corneas that peaked at 2 weeks and remained elevated at 12 weeks after surgery. MMC treatment significantly (P < 0.05) reduced corneal haze at 2 weeks and was essentially normal by 12 weeks. Imaging of SHG signals in vehicle-treated eyes showed an anterior layer of collagen forming a honeycomb network blending into a dense mat of irregularly arranged collagen fibers that overlaid normal orthogonally arranged collagen lamellae. MMC treatment showed normal collagen organization at the surface. Fibrotic tissue was associated with a high cell density and alignment of intracellular actin filaments with collagen fiber bundles. In MMC-treated eyes, an anterior acellular zone overlaid a sparsely populated stroma containing isolated and enlarged keratocytes. CONCLUSIONS Imaging of SHG signals provides a sensitive means for detection of corneal fibrosis after surface ablation and can be used to assess the effects of antifibrotic therapy on corneal healing after refractive surgery.

IGF-II and collagen expression by keratocytes during postnatal development ☆

Keratocytes produce the extensive stromal matrix of the cornea during the late embryonic and neonatal time periods. We propose to test the hypothesis that their biosynthetic activity declines during this process. Keratocytes were isolated from corneas of 6-8-week-old rabbits and corneas of 1-2-year-old cows and their ability to proliferate and synthesize collagen in serum-free media was determined. Rabbit keratocyte cultures increased 38% in DNA content after one week and deposited collagen type I and IGF-II in the media. Bovine keratocyte cultures, in contrast, did not increase in DNA or produce detectable collagen and IGF-II. Bovine keratocytes cultured in media previously conditioned by rabbit keratocytes, however, increased 56% in DNA content, and deposited collagen type I into the media. Microarray analysis of mRNA from neonatal and adult mouse keratocytes was used to confirm these differences. Compared to adult mouse keratocytes, neonatal keratocytes showed high expression levels of IGF-I, IGF-II and collagen types III and V. Since previous studies showed that IGFs stimulate bovine keratocytes to proliferate and to synthesize procollagen type I, we therefore propose that the results of this study suggests that the IGFs may play an important role in regulating early corneal growth in vivo.

Assessing ocular irritation potential using a modified ex vivo rabbit eye test.

We have evaluated the ocular irritancy potential of an unknown environmental contaminant, para-toluene sulfonic acid (pTSA), compared with that of known irritants, 5% sodium dodecyl sulfate (SDS) and 10% acetic acid (AA), using a simplified, ex vivo rabbit eye test modified to measure cytotoxicity as a mechanistic correlate to the Draize rabbit eye test. Rabbit eyes were obtained fresh within 24 hours from an abattoir and then exposed to 50 μL of test material. Eyes were then incubated intact for 3 hours or 1 day, and the corneas were removed, stained with calcein acetoxymethylester (AM)/ethidium homodimer (live/dead assay, Invitrogen Corp., Carlsbad, CA, USA), and evaluated by laser scanning confocal microscopy. The number of dead cells was then quantified and the difference was statistically compared. For corneas exposed to 1 ppm and 1% pTSA, there was no significant difference in the number of dead cells compared with water-exposed, control corneas at either 3 hours or 1 day after exposure. However, corneas exposed to 10% and 50% pTSA showed significantly increased (p < .0001) numbers of dead cells, averaging (mean ± standard deviation) 486 ± 133 and 1,052 ± 101 cells/field of view (460 × 460 μm). The level of cytotoxicity was comparable with that observed for 10% AA, which averaged 409 ± 142 cells/field of view. The data suggest that pTSA is an innocuous irritant at exposure levels environmentally encountered, but that higher concentrations (10% and above) might be considered a slight to mild irritant. We conclude that this modified ex vivo rabbit eye test using the live/dead assay may be a useful model for developing ocular irritation assays.