GAULEY, J.L.*; DEWITTE-ORR, S.J.; LEE, P.; BOONE, A; VIJAYAN, M.M.; BOLS, N.C.; University of Waterloo, Waterloo, Ontario: Response of the Rainbow Trout Macrophage Cell Line RTS11 to Lipopolysaccharide and the Influence of Temperature

Lipopolysaccharide (LPS) is in the outer leaflet of the outer membrane of gram-negative bacteria, which includes some important fish pathogens. LPS is a classic activator of macrophages, and short exposures (4h) were shown previously to enhance interleukin 1beta (IL-1beta) expression in the rainbow trout macrophage cell line, RTS11. In this study, RTS11 cultures with or without fetal bovine serum (FBS) were exposed to LPS for up to 4 weeks at 18 &degC, which is a routine growth temperature for trout cell lines, and at 28 &degC, which does not support proliferation. RTS11 cultures at a high cell density could be maintained at 18 &degC without FBS for at least 30 days with little change in cell number. When exposed to LPS under these conditions, RTS11 began to die at approximately seven days. With FBS, LPS was weakly cytotoxic to RTS11 only after 3 weeks of exposure to the highest concentration (100 &microg/ml). By contrast, RTS11 at a low cell density in FBS were stimulated to proliferate by LPS. At 28 &degC RTS11 survived for over a week in medium with FBS and synthesized heat-shock proteins (HSPs). Exposure to LPS did not change survival time at 28 &degC nor accumulation of HSPs, but did reduce induction of IL-1beta mRNA. In serum-free medium, RTS11 cells died between 3-4 days at 28 &degC and again LPS exposure did not influence survival time. Surprisingly, when RTS11 were maintained in serum-free medium for two days at 28 &degC, then shifted to 18 &degC and dosed with LPS, the cells were less susceptible to the cytotoxic actions of LPS. These results suggest that the response of rainbow trout macrophages to LPS is complex and is regulated in part by culture conditions and prior exposure to heat.