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Keywords:

  • Cornea;
  • CD34;
  • Hemopoietic stem cell;
  • Dendritic cell;
  • Leukocytes

Abstract

Previous studies have suggested that corneal stromal keratocytes express the CD34 antigen. We wished to investigate CD34 antigen expression in normal mouse cornea using dual- and triple-staining techniques. Whole-mount preparations of mouse and rat corneas were examined with confocal microscopy using single, dual, or triple immunostaining to study their morphology, phenotype, and distribution. Single-cell suspensions from normal mouse corneas were also prepared and analyzed by flow cytometry. After short-term culture of corneal stromal explants, nonadherent cells were harvested and cytospins were prepared and stained for different markers.

Combined staining for F-actin and leukocyte differentiation markers clearly showed that the corneal stroma contains a population of CD45+ resident bone marrow–derived cells, whereas most cells were CD45-F-actin+ keratocytes. A significant proportion (two thirds) of CD45+ cells in the normal corneal stroma expressed CD34+, whereas no CD45 cells (i.e., keratocytes) coexpressed CD34. In addition, CD34+ cells were CD11c and CD11b+. Fewer than 10% of the CD34+ cells also coexpressed Sca-1+, but no CD34+ cells coexpressed major histocompatibility complex (MHC) class II+. In contrast, the remaining population of CD45+CD34 cells in the corneal stroma expressed CD11b, MHC class II+ but not CD11c and were found mostly in the anterior and peripheral part of stroma. These cells are in intimate contact with corneal keratocytes, which stained only for F-actin and were negative for all leukocyte markers. Very few CD45+ cells expressed the B220 marker, suggesting a plasmacytoid dendritic cell phenotype. Flow cytometry analyses confirmed the morphometric data showing that 68% of CD45+ cells coexpress CD34 and CD11b, whereas 22% are CD11b+CD34.

We conclude that the normal mouse cornea contains two populations of bone marrow–derived leukocytes, both of which are distinct from stromal keratocytes. The larger population resembles CD34+ hemopoietic stem cells, whereas the smaller population are CD34CD11b+ MHC class II+ macrophages. A very small percentage comprises plasmacytoid dendritic cells.