Development of leucocyte cell lines for immunological research in teleost fish

White blood cells, or leucocytes, are the cell that defence the body from infections and provide disease resistance. Research into the function of leucocytes is greatly aided in mammals by the availability of many leucocyte cell lines, that allow a lot of research to be performed using culture systems without the need to harvest tissues and cells from animals. These developments have been possible in large part by the discovery of many leucocyte growth factors, that can be produced using molecular methodologies, and added to the cultures as growth supplements. In this proposal we aim to develop leucocyte cell lines for fish, due to increasing use of fish by researchers, partly as a consequence of the major expansion of aquaculture and its associated needs (eg fish health issues). The applicants are internationally renown for their work on the fish equivalents of the leucocyte growth factors, and so can produce relevant proteins to encourage leucocytes to grow in vitro. Several different approaches will be taken to generate the cell lines, and some preliminary characterisation is proposed, so that they will quickly become a valuable resource for researchers. In addition to a reduction in the numbers of fish being used, a reduction in fish suffering should also follow this development, since the cell lines will also allow preliminary in vitro testing of compounds and chemicals to see if they affect leucocyte function, before testing in live fish.

This programme aims to take advantage of our recent success in cloning fish cytokines, to develop fish leucocyte cell lines. We will produce the recombinant proteins and add to leucocyte primary cultures as growth factors to allow their growth in vitro. In addition, we will combine this treatment with antigen stimulation, by using leucocytes derived from immunised fish. In addition to morphological studies, we will undertake molecular and functional analysis to give a preliminary characterisation of the cell types that grow under these conditions and determine their cytotoxic and phagocytic activities. We will test whether the cell lines can be used to measure responses to exogenous immunostimuli such as lipopolysaccride, polyI:C and recombinant cytokines. We believe that the established cell lines will provide a permanent platform for basic research of the fish immune system, thereby reducing the need to kill fish to harvest tissues. They will also help reduce fish suffering by allowing some testing of aquaculture drugs/reagents, and the impact of some chemicals/pollutants to be carried out in vitro before in vivo studies, to establish whether they impact on leucocyte function and therefore potentially fish health and welfare.