The ontogeny of mucosal immune cells in common carp (Cyprinus carpio L.).

The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38(+) intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15(+)monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12(+) (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).

Rag expression identifies B and T cell lymphopoietic tissues during the development of common carp (Cyprinus carpio).

The generation of lymphoid cells during carp development was studied by analyzing expression of the recombination activating genes (rag) using in situ hybridization and real time quantitative PCR. These data were combined with immunohistochemistry using the mAb's WCL9 (cortical thymocytes) and WCI12 (B cells). Carp rag-1 and rag-2 showed 90 and 89% amino acid identity, respectively, to the corresponding zebrafish sequences. Rag-1 was first expressed in the thymus at 4 days post-fertilization (dpf), while both rag-1+/WCL9+ and rag-1-/WCL9- areas were distinguished from 1 week post-fertilization (wpf), suggesting early cortex/medulla differentiation. From 6 dpf, rag-1+ cells were also present cranio-lateral of the head kidney. From 1 wpf, rag-1/rag-2 was expressed in kidney (together with immunoglobulin heavy chain expression) but not in spleen, while WCI12+ cells appeared 1 week later in both organs, suggesting B cell recombination in kidney but not in spleen. Rag-1 expression exceeded rag-2 levels in thymus and in head- and trunk-kidney of juveniles, but this ratio was reversed in head- and trunk-kidney from approximately 16 wpf onwards. Rag-1/rag-2 expression was detected in thymi of animals over 1-year-old, but in kidney only at low levels, indicating life-long new formation of putative T cells but severely reduced formation of B cells in older fish.