The first appearance of Rodlet cells in carp (Cyprinus carpio L.) ontogeny and their possible roles during stress and parasite infection.

The origin and function of rodlet cells (RCs) are still a matter of discussion. Whereas the exogenous hypothesis considers them parasites, the endogenous hypothesis regards them as a genuine fish cell population with a secretory and/or leukocyte function. In order to shed more light on these questions we focused on the location and appearance of RCs during carp (Cyprinus carpio) ontogeny. Typical RCs were seen at 5days post fertilisation (dpf) between kidney and intestine, at 6dpf in the intestine and at 8dpf in both anterior and posterior kidney and in the abdominal cavity among the mesothelial cells. The RC number increased with age and after 14dpf they were also present in gills. The early appearance of the RCs during carp ontogeny support the endogenous hypothesis stating that RCs are genuine constituents of fish tissue and suggest that they are 'immune cells'. The fact that the RCs of the gills secrete their content into the surrounding water, combined with the strategic location around blood vessels in kidney and within intestinal epithelium, would also support an important role in host defense. To investigate whether RC numbers in gills and kidney are related to typical fluctuations in the physiology during stress and infection we counted their number in gills and kidney after parasite infection and stress. In the gills the number of RCs increased after infection but did not change after stress while in the kidney their number increased after stress and no significant changes were observed after infection.

Structural characterisation of a cyprinid (Cyprinus carpio L.) CRH, CRH-BP and CRH-R1, and the role of these proteins in the acute stress response.

We elucidated the structure of the principle factors regulating the initiation of the acute stress response in common carp: corticotrophin-releasing hormone (CRH), CRH-receptor 1 (CRH-R1) and CRH-binding protein (CRH-BP). Phylogenetic analyses reveal that these proteins are evolutionarily well conserved in vertebrates. CRH and CRH-BP expression are not co-localised in the same hypothalamic perikarya. On the contrary, CRH-BP expression is limited to the perimeter of the nucleus preopticus (NPO), but is abundant in other regions, including an area directly rostral from, and in close proximity to, the NPO. Despite the lack of co-expression, the nerve fibres projecting onto both the rostral pars distalis (rPD) as well as the large fibre bundles projecting onto the pars intermedia (PI) contain CRH as well as CRH-BP, suggesting that both ACTH release from the rPD as well as the release of PI melanotrope content is regulated via CRH and CRH-BP. Finally, we show via real-time quantitative PCR that expression of hypothalamic CRH and CRH-BP following a 24 h restraint significantly increases, whereas PD CRH-R1 expression decreases; this reflects desensitisation of the PD for hypothalamic CRH output. We conclude that these factors are actively involved in the regulation of acute stress responses in the teleost fish.

Ontogeny of the thymus in a teleost fish, Cyprinus carpio L.: developing thymocytes in the epithelial microenvironment.

A monoclonal antibody, WCL9, specific for membrane molecules of a thymocyte subpopulation was used to detect these cells in situ during the ontogeny of thymus. Cryo-sections revealed WCL9+ cells in the rudiment of the thymus (day 4 post fertilization); thereafter, the positive cells were observed exclusively in the cortex from the first appearance of thymic regionalization (week 4 post fertilization) until adult age. Whole-mount immunostaining of the thymus with WCL9 revealed the three-dimensional structure of the cortex by specific staining. The presence and distribution of apoptotic cells during thymus development was studied by in situ end-labelling of fragmented DNA. From week 4 post fertilization onwards, apoptotic cells were more frequently detected in the cortex than medulla, suggesting a continuous selection of thymocytes in the cortex. Ultrastructural studies confirmed the presence of numerous cortical apoptotic cells inside macrophages. Electron microscopy provided evidence for the existence of epithelial heterogeneity in the thymus. During the ontogeny, the differentiation of epithelial cells was followed from the first weeks until the juvenile age. Cell types were classified on the basis of their localization and cytological characteristics as: i) limiting epithelial cells located in subcapsular, perivascular and peritrabecular zones; ii) reticular epithelial cells situated in medullary and cortical zones; iii) nurse-like cells at the border between the cortex and medulla, iiii) Hassall's body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

Expression of MhcCyca class I and class II molecules in the early life history of the common carp (Cyprinus carpio L.)

In this study transcription of class I alpha chain (Cyca-UA), beta2-microglobulin (Cyca-B2m) and class II alpha (Cyca-DXA) and beta (Cyca-DAB) during the early stages of embryo development was investigated by semiquantitative PCR. No transcripts of the genes under investigation were detected in the unfertilized egg. The expression of the genes encoding for the class II molecules revealed to be synchronized starting at day 1, unlike those for the class I molecules. Transcription of Cyca-B2m was first detected at day 7, whereas Cyca-UA was already present on day 1. This discrepancy would suggest absence of class I molecules during early development. The transcription of the Mhc genes in lymphoid organs was well established on day 21, with the exception of the spleen. In later stages of ontogeny cell surface expression of class I molecules was studied using polyclonal antibodies to Cyca-UA and Cyca-B2m in conjunction with detection of surface Ig. In week 3-10 Cyca-B2m was found on a higher percentage of cells from pronephros, spleen and thymus compared to Cyca-UA, suggesting the use of an alternative class I alpha chain. In the thymus, unlike the other organs, this difference remained present in the adult stage. The most likely candidates are alpha chains encoded by non-classical class I genes.

Characterisation of immunoglobulin-binding leucocytes in carp (Cyprinus carpio L.).

This study demonstrates the immunoglobulin(Ig)-binding capacity of Ig-positive carp macrophages employing immunofluorescence and immunogold methods. These methods allow for the characterisation of the Ig-binding cells. After internalisation of fluorescent- or gold-labelled Ig (30 min at room temperature), most macrophages from the hindgut were able to bind added purified carp Ig, which could be demonstrated clearly with a second fluorescent or gold label. In pronephros, an important haemopoietic organ in fish, a limited number of monocyte-like cells also showed Ig binding. Pronephros macrophages and neutrophilic granulocytes appeared to be Ig-negative. The use of goat anti-mouse Ig gold particles bound by carp anti-goat antibodies revealed that, in addition to hindgut macrophages and pronephric monocyte-like cells, some lymphoid cells in both hindgut and pronephros cell suspensions were also able to bind Ig. The classic erythrocyte-antibody rosette assay resulted in a limited number of small rosettes in cell suspensions from both organs.

Differences in mucus and serum immunoglobulin of carp (Cyprinus carpio L.).

Electrophoretic analysis did not reveal clear differences between skin mucus and serum immunoglobulin (Ig) of carp. The majority of both Igs were tetrameric (+/- 760 kDa) and composed of 25 kDa light (L) chains and 70 kDa heavy (H) chains, but dimeric and monomeric forms were found as well. Monoclonal antibody (mAb) WCI 12 produced from serum Ig appeared to react with the H chain of both molecules. After immunisation of mice with purified mucus Ig, mAbs could be selected that were reactive with mucus Ig only. Two of these mAbs (WCI M1 and WCI M2) were immunoreactive with the H chain of mucus Ig and not or hardly immunoreactive with the H chain of serum Ig, indicating differences in the composition of the H chains of both molecules. Because WCI M2 appeared to recognize a carbohydrate determinant, differences seem to occur in the protein as well as carbohydrate composition of mucus and serum Ig. Flow cytometric results showed that both mAbs were reactive with the same subpopulation of WCI 12-positive B cells. Immunohistochemical reactions on cryosections also showed a limited reaction by these mAbs compared with WCI 12; only epithelium of skin and bile ducts and capillaries in the liver were strongly positive with these mAbs. The presence of mucus Ig at these locations is discussed. Our results indicate structural and functional differences between mucus and serum Ig, which may explain the mucosal immune responses reported for fish. Such a specific mucosal defense system can be very important for fish, living in a pathogen-rich environment.

The gut-associated lymphoid tissue (GALT) of carp (Cyprinus carpio L.): an immunocytochemical analysis.

The GALT of carp was studied with monoclonal antibodies reacting with carp Ig or carp leukocytes, using (dual) immunofluorescence or immunogold staining on cryosections, cytocentrifuge slides, and cell suspensions of the intestine. The intestinal epithelium contained many Ig-negative lymphoid cells and, in the hindgut, also many large Ig-positive macrophages, which appeared to bind Ig. The lamina propria contained numerous Ig-positive lymphoid cells next to Ig-negative lymphoid cells and granulocytes. Leukocytes isolated from the intestine mainly consisted of Ig-negative lymphoid cells (> 90%). With the methods used, leukocytes were poorly released from the connective tissue. Nevertheless, two types of Ig-containing cells were found: a conventional plasma cell, frequently showing Ig at its surface, and a more common smaller lymphoid cell having a narrow rim of Ig-positive cytoplasm but hardly any Ig on its surface. Many of the Ig-positive lymphoid cells observed in the lamina propria may represent these small Ig-containing cells. Isolated Ig-positive macrophages were frequently associated with B- and T-like cells. Our data strongly suggests an immunological function for the gut of carp, especially for the antigen-transporting hindgut.