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 24 hr 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 24 hr 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.

The distribution of antigen in lymphoid tissues following its injection into the anterior chamber of the rat eye.

Injection of Ag into the anterior chamber (AC) of the eye induces deviant immune responses. It has been proposed that Ag internalized by ocular APCs is presented in a tolerogenic fashion in the spleen. However, the nature and distribution of the Ag-bearing cells in the lymphoid organs remain unclear. Fluorescent-labeled Ag (dextran, BSA) injected into the AC of Lewis rats was detected in the subcapsular sinus of the right submandibular lymph nodes (LNs) and cervical LNs, the marginal zone of the spleen, and the medulla of the mesenteric LNs. In the spleen, Ag-bearing cells were CD1(+), CD11b(+), ED1(+), ED2(low), ED3(+), CD86(low), OX6(+), CD11c(-), ED5(-) and in the LNs were CD4(+), CD8(+), CD80(+), and OX41(+) suggesting these were lymphoid organ resident macrophages. These Ag-bearing macrophages were located adjacent to CD4(+) cells, CD8(+) cells, and NK cells in the LNs and spleen and to marginal zone B cells in the spleen. No interaction with gamma delta T cells was observed. The data demonstrates that Ag derived from the AC of the eye is mainly internalized by resident macrophages in the LNs and spleen which are ideally placed to interact with cells involved in the induction of deviant ocular immune responses. The extensive distribution of Ag in LNs draining the upper airway and gastrointestinal tracts, together with the phenotype of Ag-bearing cells in the lymphoid organs, suggests that Ag leaves the eye predominantly in a soluble form and implies other mechanisms of tolerance may contribute to ocular-specific immune responses.

Local retention of soluble antigen by potential antigen-presenting cells in the anterior segment of the eye.

PURPOSE: To determine the capacity of bone marrow-derived cells in the anterior segment of the eye to capture a fluorescence-labeled antigen (Ag) injected into the anterior chamber (AC). METHODS: Uveal tract and corneoscleral tissues from Lewis rats were cultured in vitro, with or without FITC-dextran (4 microg/mL final concentration), for 48 hours and examined by confocal microscopy. To investigate antigen uptake in vivo 2 microL (20 microg) of Cascade Blue-labeled dextran (CB-Dx) was injected into the right AC of Lewis rats. The density of Ag-positive cells in the iris at 1, 3, 5, or 12 days after injection was examined by in vivo video fluorescence microscopy. The distribution and phenotype of Ag-positive cells in frozen and paraffin-embedded sections of ocular tissues and in iris wholemounts from animals killed at 24 hours and day 7 were analyzed by fluorescence and confocal microscopy. RESULTS: In organ culture conditions numerous cells in the iris, ciliary body, choroid, and corneal limbus were capable of capturing fluorescence-labeled Ag. In vivo observations and microscopic examination of experimental eyes at days 1 and 7 after AC injection revealed Ag-positive cells within the iris, iridocorneal angle, the suprachoroidal space and around limbal-episcleral vessels. Ag-bearing cells in the iris express combinations of macrophage markers but rarely expressed major histocompatibility complex (MHC) class II molecules. A reduced number of Ag-bearing cells were still present in the iris at day 12. CONCLUSIONS: Potential antigen-presenting cells (APCs) in the iris and ciliary body are capable of internalizing intracameral Ag. The characteristics of these cells in the iris are consistent with a predominantly macrophage phenotype. These observations also suggest that the Ag leaving the eye through both the conventional and nonconventional aqueous outflow pathways may be captured by potential APCs in the episcleral tissues.