Abstract
Purpose: :
Dendritic cells (DCs) are efficient antigen–presenting cells (APCs) important for initiating immune responses. It was long thought that DCs resided in the periphery, but were absent from the center of normal cornea. Recent studies have identified some DCs in central cornea in ex vivo preparations. Here, we examined the distribution and migratory capability of cells expressing the DC marker CD11c in murine cornea using in vivo and ex vivo microscopy.
Methods: :
Mice expressing enhanced yellow fluorescent protein (eYFP) under the control of the CD11c promoter were used. To assess normal distribution and mobility of CD11c+ cells, unmanipulated corneas were imaged with in vivo wide–field and ex vivo confocal fluorescence microscopy. To examine response of CD11c+ cells to challenge with soluble or particulate antigen, red fluorescent ovalbumin (OVA) (50 µg) or red fluorescent microspheres (1 µm) were injected intrastromally. Control eyes were sham–injected. At various time points (starting at 6, 16, or 24h), multicolor in vivo real time or time–lapse imaging was performed to track movement of eYFP+ cells, red beads, or red OVA+ cells. Corneas were imaged at 3 frame/min for 30 min to visualize quick, or at 1 frame/h for 8h to detect slow migration. Videos were stabilized and individual cell or bead locations were tracked. Confocal image stacks were deconvolved and used to produce 3–D reconstructions.
Results: :
In unmanipulated corneas, CD11c+ cells were sparsely distributed in the central cornea (28±10 cells/mm2), but increased toward the periphery (190±26 cells/mm2). Peripheral cells appeared to have finer dendrites and slimmer cell bodies compared to those in central cornea. Confocal microscopy showed CD11c+ cells to be located primarily in the epithelium and anterior stroma centrally, but present at various depths in the periphery. Video microscopy showed that while processes of some cells appeared to move, cell bodies did not. With injection of ovalbumin or microspheres, CD11c+ cell migration was detectable when corneas were imaged repeatedly over hours but not within a 30 min period. This indicated that, in contrast to the migratory capability of neutrophils seen in other models, migration of CD11c+ DCs is relatively slow.
Conclusions: :
The presence and function of corneal DCs have important implications for a variety of physiologic and pathologic responses. Distribution of corneal CD11c+ cells as shown by in vivo and ex vivo examination is in general agreement with ex vivo studies using different techniques. Dynamic in vivo imaging studies suggest that mobility of these cells appear to be different from what we have measured of other leukocytes that migrate through the cornea.
Keywords: cornea: basic science • imaging/image analysis: non-clinical • inflammation