Steady state dendritic cells (DC) found in non-lymphoid tissue sites under normal physiologic conditions play a pivotal role in triggering T cell responses upon immune provocation. CD11b+ and CD103+ DC have received considerable attention in this regard. However, still unknown is whether such CD11b+ and CD103+ DC even exist in the ocular mucosa, and if so, what functions they have in shaping immune responses. We herein identified in the ocular mucosa of normal wild-type (WT) and Flt3-/- mice the presence of a CD11b+ DC (i.e., CD11c+ MHCII+ CD11b+ CD103- F4/80+ Sirp-a+). CD103+ DC (i.e. CD11c+ MHCII+ CD11b low CD103+ CD8a+ DEC205+ Langerin+) were also present in WT, but not in Flt3-/- mice. These CD103+ DC expressed high levels of Id2 and Flt3 mRNA; whereas CD11b+ DC expressed high Irf4, Csfr, and Cx3cr1 mRNA. Additionally, the functions of these DC differed in response to allergic immune provocation. This was assessed utilizing a previously validated model, which includes transferring specific populations of exogenous DC into the ocular mucosa of ovalbumin (OVA)/alum-primed mice. Interestingly, in such mice, topical OVA instillation following engraftment of exogenous CD11b+ DC led to dominant allergic T cell responses and clinical signs of ocular allergy relative to those engrafted with CD103+ DC. Thus, although CD11b+ and CD103+ DC are both present in the normal ocular mucosa, the CD11b+ DC subset plays a dominant role in a mouse model of ocular allergy.

The wound-healing response is critical to the outcome of refractive surgery and studying wound healing contributes to an understanding of the pathophysiology of other corneal injuries. Animal models allow research to be conducted with sufficient samples and under controlled parameters. We studied the hen to determine the healing process from clinical, biophysical, and biological standpoints after photorefractive keratectomy (PRK). PRK (ÿ6.0 diopters) was performed in hen eyes. At 3, 6, 12, 24, 48, and 72 h and 5, 7, 15, 30, and 60 days postoperatively, we studied the clinical follow-up, objective measurements of light transmission (direct transmittance), apoptosis by TUNEL assay, proliferation by immunocytochemical analysis of 5-bromo-2 0 -deoxyuridine, and expression of alpha smooth muscle actin (SMA) in myofibroblasts in the corneas. Hen corneas reepithelialize quickly. Haze developed from 5 to 60 days after surgery and was correlated with the appearance and finalization of the expression of SMA. The direct transmittance of light was low during the first 15 days and improved at 30 and 60 days. TUNEL-positive cells were observed 3 h after surgery and the numbers decreased thereafter. Epithelial proliferation began at 12 h and was greater at 48 h, while stromal cell proliferation began at 24 h and was greater at 72 h. The hen cornea is anatomically similar to the human cornea, and the manner in which it heals is a good model for studying different surgical techniques and pharmacologic assays.

PURPOSE. Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro.METHODS. An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Realtime migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (b4) expression was assessed by ICC and WB.RESULTS. Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased b4 levels were observed after adding NGF (P < 0.01).CONCLUSIONS. NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of b4 integrin.