Rosemarie C. Ganassin

ECOTOXICOLOGY AND INNATE IMMUNITY IN FISH

This review summarizes the scattered literature on the effects of toxicants on the external and internal innate immunity of fish. Insecticides, heavy metals and surfactants have been the most frequently examined toxicants, whereas dioxins, furans and polychlorinated biphenyls have been tested less frequently. Studies to date have been conducted at the levels of cells in vitro, of fish in the laboratory and microcosms, and also of fish in the field. Among innate immune parameters, phagocyte respiratory burst appears especially sensitive to toxicants. Toxicant-induced alterations in external mucous production have also been observed repeatedly. Field studies have occasionally examined changes to melano-macrophage centers, but the meaning of such changes is not clear. Advances in basic knowledge of fish innate immunity should lead to improvements in monitoring fish health and predicting the impact of toxicants on fish populations, which is a fundamental ecotoxicological goal.

Production of zebrafish germ-line chimeras from embryo cell cultures

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic–stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

SummaryA rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [3H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.

Cell and Tissue Culture

Publisher Summary The culturing, development, and monitoring requirements vary with the goals, cell type, and species under investigation, much information can be obtained from the protocols for developing specific types of fish cell cultures and for monitoring general and specific functions in fish cell cultures. This chapter describes methods for developing primary long-term hemopoietic cultures from fish. An active hemopoietic culture continuously produces blood cells for a period of up to six months. The influence of various additions to the basic culture medium (for example, toxicants, growth factors, and cytokines) provides information about the factors that perturb blood cell formation. The methods of measuring general and specific cell parameters useful in both basic research and aquatic toxicology are outlined in the chapter. These are cell viability and the induction of 7-ethoxyresorufin-o-deethylase (EROD) activity in fish cell cultures. The results of cell viability assays can be measured rapidly and inexpensively in microwell cultures with a multiwell fluorometric plate reader, improving the utility of fish cell cultures in many disciplines but especially in toxicology. The species used is rainbow trout (Oncorhynchus mykiss) because it is the most commonly used species in fish research.

Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitzs L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.

A DNA fluorometric assay for measuring fish cell proliferation in microplates with different well sizes

The use of a DNA-binding dye, bisbenzimidazole (H33258), and a microplate fluorometer, CytoFluor 2350, was optimized to measure cell number in Chinook salmon embryo cell cultures (CHSE-214). The uniformity of cell homogenates, which were prepared prior to staining, was evaluated by area scan, which consists of multiple measurements over the total well area. Disruption in 0.01% SDS produced a relatively uniform homogenate and a low background. Homogenates were stained with H33258 at 1 to 10 µg/ml. Linear relationships between cell numbers and fluorescence units were established in 96 well plates, which were read only by standard scan, and in 6, 12, 24 and 48 well plates, which were read by area and standard scan. Area scan provided better relationships. These methods were used to show that in L-15 medium CHSE-214 were able to attach, retain viability, and proliferate with very little exogenous calcium.

Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix

A technique for the clonal culture of rainbow trout leukocytes in a methylcellulose matrix can be used to identify growth factors and other substances affecting cell proliferation and development in fish. Methylcellulose supports colony formation by rainbow trout leukocytes isolated from the major hemopoietic organ, the pronephros. The addition of rainbow trout serum dramatically increased the number of colonies formed, scored by counting colonies. As an alternative measure of cell proliferation, 3H-thymidine incorporation by cells can be easily measured in methylcellulose cultures. This method requires only small amounts of test substances, is rapid, and is superior for assessing the growth-stimulating ability of some substances, such as bacterial lipopolysaccharide, which stimulated growth but not the formation of discreet colonies by rainbow trout cells.

Effect of Exposure to Various Sites within Hamilton Harbour on Oncorhynchus mykiss Pronephros Macrophage Function and B Cell Numbers

Our objective was to assess whether or not exposure to sites within Hamilton Harbour known to be highly contaminated with polycyclic aromatic hydrocarbons (PAHs), heavy metals, and sewage treatment plant (STP) effluent could affect macrophage function and B-cell numbers in fish. Caged rainbow trout (Oncorhynchus mykiss) were sampled after 7, 14, and 21 days of exposure to five harbor sites, plus a Lake Ontario reference site. Four of the harbor sites were selected for their proximity to either highly contaminated sediments or industrial and municipal discharges; the fifth site was selected as a harbor reference site. Pronephros leukocytes from fish at the six sites were evaluated for phagocytic activity, oxidative burst, and the number of surface immunoglobulin-positive B cells. Although none of the immune parameters and B-cell numbers measured from fish that were caged at the contaminated harbor sites were significantly different from the harbor reference site, they were significantly different from the Lake Ontario reference site. Fish showed a greater reduction in pronephros leukocyte phagocytic activity over the course of the study when compared to the Lake Ontario reference fish; pronephros leukocyte oxidative burst was also reduced at two of the harbor sites. B cell counts did not change throughout the duration of the study, although overall counts were lower at two of the harbor sites when compared to the Lake Ontario site. The results indicate that macrophage function and B-cell numbers were altered in fish by exposure to various sites in Hamilton Harbour. The possible immunomodulatory roles of chemical and physical parameters at those sites are discussed.

Use of Cultured Primordial Germ Cells for Production of Transgenic Fish

Due to its favorable characteristics, the zebrafish is a popular model of vertebrate development. However, one deficiency of the zebrafish model system is the lack of methods for cell-mediated gene transfer and targeted mutagenesis. In mice, cell-mediated gene transfer is accomplished through the use of embryonic stem (ES) cell cultures and provides the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. ES cells possessing a targeted mutation are selected in culture and transferred to a host embryo. Transgenic mice possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of the chimeric embryo. In zebrafish, embryo cell cultures have been derived that exhibit in vitro characteristics of ES cells but successful contribution of the cells to the germ cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures, maintained in the presence of cells from a rainbow trout spleen cell line, RTS34st, are able to produce germ line chimeras when introduced into a host embryo. Zebrafish embryo cells co-cultured with RTS34st cells or their conditioned medium continue to possess mRNA encoding the primordial germ cell marker, vasa, for more than 30 days. In the absence of RTS34st cells or conditioned medium the vasa mRNA disappeared by five days in culture. The spleen cells also inhibited the embryo cell cultures from differentiating into melanocytes and neuronal cell types. The influence of the RTS34st cells on the zebrafish embryo cell cultures indicate that the splenic stromal cell line will be a valuable tool in the application of cell-mediated gene transfer and targeted gene inactivation technology to zebrafish.