An intravital and confocal microscopic study of the distribution of intracameral antigen in the aqueous outflow pathways and limbus of the rat eye

In a previous investigation into the fate of fluorescently labelled antigen (Ag) injected into the anterior chamber (AC) of the rat eye, a large number of Ag ⁺ cells were noted in the conventional and non-conventional aqueous humour outflow pathways together with the external limbus. The aim of this study was to investigate the precise distribution and phenotype of these cells and compare their ability to capture fluorescent-labelled protein (bovine serum albumin, BSA, and ovalbumin, OVA) and polysaccharides (dextran, Dx) injected into the AC. The density of Ag ⁺ cells in the iris and limbus was investigated using in vivo video fluorescence microscopy 24hr post-injection. The distribution and phenotype of Ag ⁺ cells in ocular tissues was analysed by confocal microscopy of frozen sections and in iris and corneoscleral/limbal wholemounts from animals sacrificed 24hr post injection. The general distribution of labelled Ag was equivalent in OVA, BSA and Dx injected animals. Antigen-bearing cells were observed within the iris, iridocorneal angle, pre-equatorial choroid and around limbal/episcleral vessels. Localization of Ag ⁺ cells and free Ag in the anterior segment suggests that substances of these molecular weights (40-70 kDa) leave the eye through the conventional and non-conventional aqueous outflow pathways. The cells that internalized BSA, OVA or Dx in ocular tissues were of a similar phenotype, namely, ED1 ⁺, ED2 ⁺, occasionally ED3 ⁺ and predominantly MHC class II ^-, thus suggesting that they are of the macrophage phenotype. However, a few Ag ⁺ MHC class II ⁺ dendriform cells (putative DC) were also observed in the iris, trabecular meshwork, choroid and episclera. In conclusion our data reveal that the majority of intracamerally injected soluble Ag retained in the eye is taken up by resident macrophages not only in the iris but in all tissues lining the AC of the eye.