Transforming Growth Factor-Beta in Teleost Fish: Development of a Quantitative Assay for MRNA Expression and Application to a Field Study of Fish Health
Transforming growth factors (TGF)- β are cytokines with diverse functions affecting cell growth and differentiation, extracellular matrix regulation, wound healing and immune function. TGF-β immunoregulatory properties are primarily immunosuppressive, and increased levels of TGF-β have been found in several disease states associated with immunosuppression in mammals, including different forms of malignancy, chronic degenerative diseases, and AIDS. The ability to measure differences in TGF- β production in fish could provide a valuable tool for evaluating the impact of environmental contaminants and infectious agents on fish immune systems.
A transforming growth factor (TGF)-β was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF- β (57.3% and 78.6% identity with precursor and active protein respectively) and rat TGF- β (41.1% and 68.8% identity with precursor and active protein respectively). Consensus primers were designed which specifically amplify by polymerase chain reaction (PCR) a TGF-β segment from at least 11 species of teleost fish comprising 8 taxonomic families in 5 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-β mRNA expression in teleost fish. Higher levels of TGF-β mRNA expression was detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.
During summer of 1998, the RT-qcPCR assay for TGF- β was incorporated into a larger study of fish health from 4 tributaries of Chesapeake Bay. White perch (Morone americana) were sampled from the Pocomoke, Wicomico, Choptank and Back Rivers in June, August and October. Splenic mononuclear cell TGF-β transcription levels were initially highest in perch from the Back River, which flows through an industrial area of Baltimore, than from the 3 eastern shore rivers. However, levels from Back River fish remained steady throughout the sampling period, while significant increases to values greater than those from the Back River were noted from the other 3 rivers in August and October, coincident with the appearance of cutaneous ulcerative lesions in menhaden (Brevoortia tyrannus) from those waters. Anterior kidney macrophage bacteriocidal activity declined on a population basis inversely to the rise in TGF- β expression. While elevated TGF-β expression and depressed macrophage bacteriocidal activity did not correlate on an individual fish basis, these findings together suggest seasonal immune system modulation of white perch from Chesapeake Bay eastern shore tributaries.
We thank Tedd Childers for technical assistance, and Larry Piper and Craig Weedon of the Maryland Department of Natural Resources for fish collection. Financial support came from an EPA Science to Achieve Results Fellowship, an NCSU CVM State Research Support grant, and the USGS INATURES Program.