Both eicosanoid generation and the complement system have long evolutionary histories predating the emergence of the vertebrates over 500myr ago. This study investigated the interplay between these two systems in an example of a bony fish, the rainbow trout (Oncorhynchus mykiss). Specifically, it examined whether purified complement fragments including C3a-1 and zymosan-activated serum, stimulate the biosynthesis of any of these eicosanoids by trout macrophages. Incubation of macrophages with zymosan pre-incubated with normal trout serum resulted in the phagocytosis of such particles and the generation of both intra- and extra-cellularly located lipoxygenase and cyclooxygenase products. Both eicosanoid generation and phagocytosis levels were significantly elevated following incubation of zymosan in trout serum in comparison with heat-inactivated (60°C for 30min) trout serum and saline alone. A combined mass spectrometry/high performance liquid chromatography approach was employed to conclusively demonstrate the presence of the cyclooxygenase product, prostaglandin E (PGE) in the culture supernatants of ionophore-challenged macrophages. Incubation of trout macrophages with zymosan-activated trout serum (i.e. no zymosan present) failed to stimulate PGE generation. Similarly, incubation of these cells for up to 60min with C3a-1 (4 or 50nM) failed to generate significant amounts of PGE or lipoxygenase products such as leukotriene B4/5or lipoxin A4/5. Longer term (6 & 24h) incubation of macrophages with C3a-1 (4nM) resulted in a time dependent increase in the generation of PGE but not leukotriene B in culture supernatants. No conclusive evidence that the increase in PGE generation was caused by changes in the expression of either cyclooxygenase-1 or -2 was found.
Bibliographical noteE.L.M. was funded by a BBSRC Special Studentship and J.W.H was supported by BBSRC grant 1/S20314. The work in the laboratory of J.O.S was funded by the National Science Foundation (NSF-MCB-0719599). We are grateful to Dr Ed Pope (Swansea University) for advice on statistical analysis.
- Rainbow trout