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Tomas Blanco, Matthew Kan, Michael Gunn, Daniel Saban; Cornea Intravital Multiphoton Visualization of the Resident Mononuclear Phagocyte Network in Allergy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1287.
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The cornea houses an extensive network of resident mononuclear phagocytes, including macrophages, Langerhan’s cells, langerin+ dendritic cells (DC) and CD11b+ DC. Their function(s), particularly independent of recruited inflammatory monocytes, is poorly understood. A novel mouse line was established with a CX3CR1-cre x ROSA26floxSTOPfloxGFP reporter system, wherein all progeny derived from the macrophage-dendritic cell precursor (MDP) lineage permanently express eGFP. We examined these corneas via intravital multiphoton microscopy in a model of ocular allergy, previously shown by our group to have corneal manifestations
Transgenic mice were used and compared with commercially available knock in CX3CR1 GFP/+. Mice were immunized systemically with OVA (100 ug) + pertussis toxin (300 ng) + aluminum hydroxide (1 mg), or left naïve as a control. After 2 wk, mice were challenged with an eye drop of Texas Red-conjugated (250 ug OVA). Microscopy was performed before and at various indicated time-points after OVA challenge. A multiphoton microscope (900 nm emission) was used at < 5% of the laser power. 3 separate (high efficiency non-descanned) detectors in the epi position were used for 2nd harmonic generation, GFP, and Texas Red.
Before challenge, GFP+ cells were detectable within the sub-basal plexus, and seemed to be interconnected via GFP+ membrane nanotubules (MNT). Within 1-3 hrs post challenge in immunized mice, these sub-basal cells were found extending their processes and making contact with OVA at the surface. Other morphologically distinct GFP+ OVA+ cells appeared, which were intraepithelial (i.e. anterior to the sub-basal plexus) and not associated with MNT. In the stroma, GFP+ MNTs were not detectable pre-challenge. GFP+ cells became OVA+ by 6-12 hrs post challenge. This occurred despite the observation that ‘free form’ OVA seemingly could not penetrate the ocular surface. Interestingly, however, GFP+ MNT became visible in the stroma and appeared to extend upward toward the sub-basal plexus. All GFP+ cells were largely undetectable in and around 24 hours, except for infiltrated intraepithelial cells.
These data provide evidence to refute the notion that resident mononuclear phagocytes in the cornea are immunologically inert, as well as direct evidence to suggest that such cells are interconnected and may contribute to shaping an adaptive immune response.
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