Epifluorescence Intravital Microscopy of Murine Corneal Dendritic Cells

Abstract

Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study the authors examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. The authors used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess the distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the responses of resident central and peripheral corneal DCs to silver nitrate injury, lipopolysaccharide, microspheres, and tumor necrosis factor (TNF-alpha). In some experiments, TNF-alpha injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently reinjected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and were denser in the periphery, with epithelial-level DCs extending into the epithelium. Videomicroscopy showed that though cell processes were in continuous movement, cells generally did not migrate. Within the first 6 hours after stimulation, neither central nor peripheral corneal DCs exhibited significant lateral migration, but central corneal DCs assumed extreme morphologic changes. An increased number of DCs in the TNF-alpha-stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-alpha injection are more likely to respond to a secondary insult with lateral migration.

Cornea-Infiltrating and Lymph Node Dendritic Cells Contribute to CD4+ T Cell Expansion after Herpes Simplex Virus-1 Ocular Infection

“…Normal corneas are armed with stratified networks of resident DC and macrophages, but the functional characteristics of these cells remain largely undefined. For instance, the ability of the resident corneal DC to migrate from the cornea to the DLN remains controversial, particularly in the setting of corneal infection (25, 26). We and others have proposed that resident corneal DC take up viral antigens following HSV-1 corneal infections and transport them to the DLN for either direct presentation to naïve T cells or for cross presentation by DLN resident DC.…”

Dendritic Cell–Epithelium Interplay Is a Determinant Factor for Corneal Epithelial Wound Repair

“…11 These cells generally do not migrate laterally in unstimulated corneas or in corneas stimulated in the central region with silver nitrate burn injury, Escherichia coli lipopolysaccharide, or microspheres, all considered point injuries.…”

“…[7][8][9][10] In corneal epithelium stratified with five to seven layers of cells, DCs residing at the basal epithelial layer are more numerous in the peripheral than in the central cornea. 11 Some of the DCs at the central cornea insert processes between epithelial cells, and these processes might sample antigens from the environment. [11][12][13] Emerging studies 14,15 indicated a critical role for DCs in ocular inflammation and infection, especially HIV-1 keratitis.…”

“…11 Some of the DCs at the central cornea insert processes between epithelial cells, and these processes might sample antigens from the environment. [11][12][13] Emerging studies 14,15 indicated a critical role for DCs in ocular inflammation and infection, especially HIV-1 keratitis. Two recent studies 11,13 using epithelial point-injury and pro-inflammatory cytokines as stimuli reveal that DCs responded to these stimuli by changing cell orientation or migrating toward the site of stimulation.…”

“…[11][12][13] Emerging studies 14,15 indicated a critical role for DCs in ocular inflammation and infection, especially HIV-1 keratitis. Two recent studies 11,13 using epithelial point-injury and pro-inflammatory cytokines as stimuli reveal that DCs responded to these stimuli by changing cell orientation or migrating toward the site of stimulation. Although a role in maintaining epithelial homeostasis has been suggested, whether DCs are involved in epithelial wound healing and tissue repair has not been studied.…”