Purpose: Recent investigations have revealed that populations of macrophages and dendritic cells (DCs) are present in the stroma and epithelium of the cornea, although the precise phenotype and distribution are still controversial. CX(3)CR1, the sole receptor for the chemokine fractalkine, is expressed by these monocyte-derived cells. Transgenic CX(3)CR1(GFP) mice, in which either one (heterozygous) or both (homozygous) copies of the CX(3)CR1 gene were replaced by enhanced green fluorescent protein (eGFP), were used to characterize monocyte-derived cells in the mouse cornea and to determine whether the expression of this receptor influences the recruitment of these cells into the normal cornea.
Methods: Wholemount corneas were immunostained with anti-leukocyte antibodies to the phenotypic markers major histocompatibility complex (MHC) class II, CD169, CD68, CD11b, and CD45 and analyzed by epifluorescence and confocal microscopy. The density of intraepithelial MHC class II(+) cells was quantified in wild-type, CX(3)CR1(+/GFP) heterozygous, CX(3)CR1(GFP/GFP) homozygous, and CX(3)CR1-knockout mice.
Results: There was a significant reduction in the number of MHC class II(+) cells (putative DCs) in the corneal epithelium of CX(3)CR1-deficient mice (P < 0.009) compared with wild-type mice, and the few cells that were present did not possess classic dendriform morphology. No GFP(+) MHC class II(-) cells were noted in the epithelium. Dual immunostaining of corneas in both heterozygous and homozygous (CX(3)CR1-deficient) mice revealed GFP(+) cells with a more pleomorphic morphology throughout the entire corneal stroma that were CD11b(+) CD169(+), and had variable degrees of expression of CD68 andMHC class II. The immunophenotype and morphology of these intrastromal cells is strongly indicative of a macrophage phenotype.
Conclusions: This study has identified a role for CX(3)CR1 in the normal recruitment of MHC class II(+) putative DCs into the corneal epithelium and establishes a model for investigating monocyte-derived cells and fractalkine/CX(3)CR1 interactions during corneal disease.