Cytokines and tissues 357

XenoDus lymphocytes. Conversely, XenoDus TCGF is without any activity on IL-2 dependent mouse T cell lines. We have been unable to immunoprecipitate Xenopus TCGF-activity from Xenopus T cell SNs with a panel of monoclonal anti-human IL-2 antibodies that could deplete IL-2 activity from rIL-2 containing solutions. Flow cytometric analyses of propidiumiodide-stained acid-washed or non- acid-washed splenic PHAblasts (gating on light scatter and on red fluorescence) and of paraformaldehyde-fixed splenic PHA blasts or thymocytes, have revealed that only dead cells stained positive with a commercially-supplied fluoresceinated anti-human IL-2 receptor monoclonal antibody (anti-CD-25 from Becton-Dickinson). Comparably elevated fluorescence of fixed thymocytes stained with an anti XenoDus IgM monoclonal antibody was also obtained with fixed but not with live cells. Finally, we have been unable to immunoprecipitate (lactoperoxidase method) any cell surface molecules resembling an IL-2 receptor from XenoDus blasts with anti-CD-25, although this anti-Tac reagent has allowed us to immunoprecipitate a 55kDa molecule (assumed to be the IL-2 receptor beta chain) from PHA-activated human lymphoblasts. In summary, mitogen-stimulated or alloantigen-stimulated frog T cells produce molecules with IL-2-1ike activity. We have been unable to demonstrate cross reactivity of mammalian IL-2 and frog TCGF at the functional and protein levels. In addition, we have been unable to provide any data to substantiate claims that an anti-Tac antibody recognizes functionally significant (i.e. IL-2 receptor- associated) epitopes on the surface of Xenopus lymphocytes.

2.4 THE PRODUCTION OF A MACROPHAGE ACTIVATING FACTOR FROM RAINBOW TROUT LEUCOCYTES

Susan Graham and C.J. Secombes, Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, UK.

Rainbow trout (Salmo qairdneri) head kidney and blood leucocytes are capable of secreting a soluble macrophage activating factor (MAF) after stimulation with Con A. The presence of PMA as a co- stimulant increases the production of MAF. Both respiratory burst activity (NBT reduction and H202 production) and bactericidal activity against the fish pathogen Aeromonas salmonicida are enhanced after incubation of macrophages with the MAF-containing supernatants for 48-72hr. Resident (head kidney) and elicited macrophages respond equally well to MAF-containing supernatants collected 48hr after stimulation with Con A, but there is a suggestion that elicited macrophages are less sensitive to MAF using 24-hr supernatants. The target culture period before addition of MAF does not affect their responsiveness, but a continuous presence of MAF is necessary for maximal stimulation. Macrophage-depletion studies show that accessory cells are necessary for MAF production. MAF activity is inactivated at temperatures above 50°C and at pHs below 5, and can withstand four freeze/thaw cycles before there is any significant loss of activity.