Willem B. van Muiswinkel

Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance?

Two carp tumor necrosis factor alpha (TNFalpha) genes have been cloned and sequenced. Both TNF1 and TNF2 sequences have several polymorphisms in the 3 UTR and TNF2 has a polymorphism in the coding sequence. Lipopolysaccharide and the protozoan blood flagellate Trypanoplasma borreli induced expression of TNFalpha in carp head kidney phagocytes when added in vitro. Differential expression was observed, with TNF2 being higher expressed than TNF1. We used the TNFalpha-specific inhibitor pentoxifylline to demonstrate the involvement of carp TNFalpha in the induction of nitric oxide and in the stimulation of cell proliferation. In addition, two carp lines differing in their resistance to T. borreli were typed for the TNF2 polymorphism and association between one isoform and resistance was found.

Neuroendocrine-immune interactions in fish: a role for interleukin-1

Bi-directional communication between the hypothalamus-pituitary-adrenal (HPA)-axis and the sympathetic nervous system with the immune system is crucial to ensure homeostasis. Shared use of ligands and especially receptors forms a key component of this bi-directional interaction. Glucocorticoids (GC), the major end products of the HPA-axis differentially modulate immune function. Cytokines, especially interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), ensure immune signalling to the neuroendocrine system. In addition, hormones from leukocyte origin such as corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and beta-endorphin, as well as centrally synthesised and secreted cytokines, contribute to the communication network. In teleost fish cortisol is the major product of the hypothalamus-pituitary-interrenal (HPI)-axis which is the teleost equivalent of the HPA-axis. Moderate and substantial increases in cortisol during stressful circumstances negatively affect B-lymphocytes, whereas rescue of neutrophilic granulocytes may support innate immunity. Recent elucidation of lower vertebrate cytokine sequences has facilitated research into neuroendocrine-immune interactions in teleosts and the first evidence for a significant function of interleukin-1 in the bi-directional communication is discussed.

Daily handling stress reduces resistance of carp to Trypanoplasma borreli: in vitro modulatory effects of cortisol on leukocyte function and apoptosis.

Carp subjected to daily handling stress were much more susceptible to Trypanoplasma borreli infection than control fish. In a search for the cellular mechanisms involved, it was observed that cortisol suppressed T. borreli-induced expression of interleukin-1beta, tumor necrosis factor-alpha, serum amyloid A and inducible nitric oxide synthase. An NF-kappaB-inhibitor could replicate cortisol-induced apoptosis of activated peripheral blood leukocytes. In contrast, although this NF-kappaB-inhibitor induced apoptosis of neutrophilic granulocytes, cortisol prevented apoptosis of these cells, suggesting the latter process to be NF-kappaB-independent. Carp leukocytes, upon induction of apoptosis, exhibit a number of sequential metabolic alterations. First, the mitochondrial transmembrane potential (DeltaPsi(m)) is disrupted and glutathione levels are depleted, followed by exposure of phosphatidylserine on the outer cell membrane. In vitro, cortisol could inhibit NO production induced by low concentrations of lipopolysaccharide (LPS), but remarkably, enhanced NO production induced by high concentrations of LPS. However, no differences in NO production were observed in stressed versus non-stressed infected carp.

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) Hassalls body-like structures localized in the medulla. This study could suggest the occurrence of a wide range of lympho-epithelial interactions throughout thymocytes differentiation.

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 3–4xa0dpf, 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 7xa0dpf, 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 (2xa0wpf).

A history of fish immunology and vaccination I. The early days.

This historic review describes the people that were involved in studying some aspect of fish immunology and vaccination from as early as 1854. Between 1850 and 1940, most scientists were looking at fish from the angle of comparative anatomy, embryology, physiology, taxonomy and fish diseases. Most publications from this early period are describing the morphology of blood cells and hemopoietic or lymphoid organs. The first publications on specific immune responses and vaccination of fish were found in the period 1935-1938. However, the immune mechanisms behind protective immunization were largely unknown in those days. In the period after 1940, the first researchers can be found devoting their whole career to fish immunology. This paper has been organized largely by individuals and not so much by accomplishments. It is not the intent of this review to evaluate the scientific merit of the work discussed, but to provide the reader with information that was - at least in part - lost to the scientific community. Publications from before 1940 or in languages other than English (e.g. Russian) are usually not found by todays database searches on the Internet.

A short history of research on immunity to infectious diseases in fish

This review describes the history of research on immunity to infectious diseases of fish in the period between 1965 and today. Special attention is paid to those studies, which are dealing with the interaction between immune system and invading pathogens in bony fish. Moreover, additional biographic information will be provided of people involved. In the 1960s and 1970s the focus of most studies was on humoral (Ig, B-cell) responses. Thorough studies on specific cellular (T-cell) responses and innate immunity (lectins, lysozyme, interferon, phagocytic cells) became available later. In the period between 1980 and today an overwhelming amount of data on regulation (e.g. cell cooperation, cytokines) and cell surface receptors (e.g. T-cell receptor; MHC) was published. It became also clear, that innate responses were often interacting with the acquired immune responses. Fish turned out to be vertebrates like all others with a sophisticated immune system showing specificity and memory. These basic data on the immune system could be applied in vaccination or in selection of disease resistant fish. Successful vaccines against bacterial diseases became available in the 1970s and 1980s. Effective anti-viral vaccines appeared from the 1980s onwards. There is no doubt, that Fish Immunology has become a flourishing science by the end of the 20th century and has contributed to our understanding of fish diseases as well as the success of aquaculture.

Different capacities of carp leukocytes to encounter nitric oxide-mediated stress: a role for the intracellular reduced glutathione pool

Carp head kidney (HK) phagocytes can be stimulated by lipopolysaccharide (LPS) to produce nitric oxide (NO). High production of NO can suppress the carp immune system. Carp peripheral blood leukocytes (PBL) are highly susceptible but HK phagocytes are relatively resistant to the immunosuppressive effects of NO. This study demonstrates that the antioxidant glutathione plays an important role in the protection against nitrosative stress. Carp HK phagocytes, especially the neutrophilic granulocytes, contain higher levels of glutathione than PBL. Moreover, freshly isolated carp neutrophilic granulocytes have higher mRNA levels than PBL of glucose-6-phosphate dehydrogenase (G6PD), manganese superoxide dismutase (MnSOD) and gamma-glutamylcysteine synthetase (gamma-GCS). Since these molecules are part of the glutathione redox cycle, neutrophilic granulocytes have a higher capacity than PBL to maintain glutathione in a reduced state following nitrosative stress. When stimulated with LPS, neutrophilic granulocytes upregulate the expression of G6PD, MnSOD and gamma-GCS.

Analysis of lymphocyte heterogeneity in carp, Cyprinus carpio L., using monoclonal antibodies

Abstract Several in vivo and in vitro studies on functional aspects of fish lymphocytes have led to the assumption that lymphoid cell populations analogous to T- and B-cells in higher vertebrates are also present in teleosts. However, formal proof of such heterogeneity based on structure-function correlations is lacking. In this report, data on the reactivity of a number of monoclonal antibodies made against thymocytes or serum immunoglobulin of carp (Cyprinus carpio L.) are reviewed. The data suggest that the use of monoclonal antibodies will provide powerful tools with which to identify and separate structurally heterogeneous populations of fish lymphocytes so that their relative roles in the defense system of these vertebrates can be explored and exploited.