Abstract

Fish immunology is receiving increasing attention as fish assume greater importance as models in environmental toxicology, and as economically significant diseases are encountered in aquaculture. The study of fish immunology is hampered by a paucity of specific reagents, including those for detecting cytokines. Cytokines, secreted regulatory proteins and glycoproteins, mediate cellular interactions in an immune response. They include the interleukins, interferons, tumor necrosis factors, and transforming growth factors. The quantity and balance of cytokines produced can determine the type and efficacy of an immune response. Reverse transcription-quantitative competitive polymerase chain reaction (RT-qc PCR) with primers derived from conserved regions of known cytokine sequences can be used to quantify cytokine mRNA in diverse species for which specific reagents are not available. Our general hypotheses are that cytokines homologous to those in mammals are present in fish, and that measuring cytokine production will provide a tool for assessing immune status in a range of fish species. Research is focused on transforming growth factor beta (TGF-beta), a cytokine which plays an important role in immunity (being primarily immunosuppressive), development, cell proliferation and carcinogenesis. The ability to monitor TGF-beta production could provide a means to detect dysfunctional responses to environmental stressors prior to the development of overt infectious disease, developmental abnormalities, or neoplasia.

Consensus primers derived from mammalian cytokine sequences were used for RT-PCR of hybrid striped bass (M. saxatilis x M Chrysops) anterior kidney mononuclear cell mRNA. Multiple amplification products were sequenced to determine specificity. A 416 bp RT-PCR product was cloned into a PGEM-T vector and transfected into JM109 cells. Sequencing of the plasmid insert revealed significant homology to TGF-beta precursors. Rapid amplification of cDNA ends (RACE) produced an overlapping 580 bp 3' RACE product that contains coding sequences for the entire mature active protein. This segment translates to a 112 amino acid polypeptide with 70% identity with rat TGF-beta, and 79% identity with rainbow trout TGF-beta. Acquisition of the 5' RACE product is being pursued to allow PCR amplification and isolation of a full-length TGF-beta clone. Subsequent aims are 1) measure TGF-beta mRNA from fish by RT-qcPCR and characterize tissue distribution; 2) demonstrate changes in TGF-beta mRNA expression in response to immunomodulating agents; and 3) determine the applicability of RT-qcPCR to quantify TGF-beta mRNA in multiple fish species.

Acknowledgements

The presenting author thanks IAAAM for a student travel award.