Capture of intracamerally placed Ag by resident macrophages of the spleen and of the cervical and mesenteric lymph nodes.

Abstract

Abstract: : Purpose: It has been proposed that immune tolerance induced by injection of antigen (Ag) into the anterior chamber (AC) of the eye depends on the migration of ocular antigen presenting cells from the eye to the spleen and other lymphoid organs where they deliver a tolerogenic Ag–specific signal. However, the distribution of Ag⁺ cells in the lymphoid organs following an injection into the AC of the eye remains unclear. Methods: Twenty–four hours after intracameral injection of cascade blue–labelled Dextran (CB–Dx, 70kD) into the right eye of 8–11 week–old Lewis female rats, animals were perfusion fixed and secondary lymphoid organs were collected. The phenotype of Ag⁺ cells in lymphoid tissues was determined by immunofluorescence using a range of monoclonal antibodies obtained from Serotec specific for macrophages (ED1, ED2, ED3), Dendritic cells (ED5, OX6, OX62, CD11c), T cells (TCR, CD4, CD8), NK cells (CD161) and B cells (IgM, CD45RA, RLN–9D3). Following immunostaining, tissues were analyzed by confocal microscopy. Results: Following AC injection, Ag–bearing cells were detected in the subcapsular sinus of the right submandibular and cervical lymph nodes, the marginal zone of the spleen and medulla of mesenteric lymph nodes. In the spleen and lymph nodes Ag was predominantly found within resident macrophages (CD11b⁺, ED1⁺, ED2^low, ED3⁺, CD86^low, OX6⁺, CD11c^–, ED5^–). In the lymph nodes Ag–bearing subcapsular sinus macrophages and medullar macrophages expressed CD4, CD8, CD80 and OX41. In addition a few OX62⁺Ag⁺ putative dendritic cells were observed. In all the lymphoid organs tested Ag⁺ cells expressed CD1 and were located adjacent to CD4⁺ cells, CD8⁺ cells and NK cells and to marginal zone B cells in the spleen. Conclusion: Our data supports the hypothesis that the majority of Ag injected in the AC travels from the eye in a soluble form rather than associated with ocular DCs and that Ag is internalized by marginal zone macrophages in the spleen, subcapsular sinus macrophages in the lymph nodes and surprisingly in subcapsular sinus and medullary macrophages in the MLN. These cells may be involved in the specific ocular immune responses and in the induction of ACAID. Presence of Ag in the MLN strongly suggests Ag reaches the gastro–intestinal tract and implies that oral tolerance might also play a role in the deviant ocular immune responses observed after an intracameral injection.

Keywords: ACAID • antigen presentation/processing • immunomodulation/immunoregulation