Studies on the activation of rainbow trout (Salmo gairdneri) macrophages and the characterization of a macrophage activating factor
Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.