Generation and characterisation of monoclonal antibodies against rainbow trout, Oncorhynchus mykiss, leucocytes.

Monoclonal antibodies were raised against head kidney macrophages of the rainbow trout, Oncorhynchus mykiss. Despite the establishment of a significant number of different hybridoma clones, none of these released antibody specific for determinants only found on macrophages. Instead, all the monoclonal antibodies generated reacted with lymphocytes, granulocytes, and monocytes/macrophages, although thrombocytes (the platelet equivalents in fish) and erythrocytes were not recognized by these antibodies. Western blotting of solubilised macrophages revealed that two of the hybridoma lines, designated 21G6 and 21F11, reacted with at least five proteins of 80, 104, 110, 140, and > 170 kDa. Immunocytochemistry was performed on histological sections of trout alimentary canal, gill, liver, spleen, and haemopoietic head kidney using antibodies from several of the hybridoma lines, and all of these showed a similar pattern of reactivity in each tissue. In the alimentary canal, for example, immunoreactive material was found in the eosinophilic granular cells, blood vessel margins, mucus in the lumen, and in the columnar epithelial cells. In the gills, epithelial cells and blood vessels also showed intense immunoreactive products, while in the liver, such reactivity was localised in the sinusoids and adherent macrophages. Both the spleen and head kidney had largely homogenous immunoreactivity.

Characterisation of the binding of leukotriene B4 to macrophages of the rainbow trout Oncorhynchus mykiss.

The binding of leukotriene B4 (LTB4) to macrophages from the head kidney of the rainbow trout Oncorhynchus mykiss was measured. Binding of [3H]LTB4 achieved a steady state after approximately 30 min of incubation and was 30% reversible in the presence of a minimum of 1000-fold excess of LTB4. Scatchard analysis of the kinetics of LTB4 binding over a range of [3H]LTB4 concentrations indicated the existence of only a single class of receptor with a dissociation constant, KD, of 0.14 nmol l-1 and a maximum receptor density, Bmax, of approximately 17,800 sites per macrophage. The LTB4 receptor antagonist LY223982 was ineffective in inhibiting the binding of [3H]LTB4 to trout macrophages, although another receptor antagonist, LTB4-dimethylamide, displaced a maximum of 25% of the total binding. LTB5 was equally effective as LTB4 at displacing [3H]LTB4, while other eicosanoids tested were without significant effect. It is suggested that the putative receptors for LTB4 on trout macrophages are similar to the high-affinity receptors for this compound reported to occur on mammalian granulocytes, although any structural similarities of the binding sites await further investigation.

Eicosanoid generating capacities of different tissues from the rainbow trout, Oncorhynchus mykiss.

The eicosanoid generating potential of the brain, gills, skin, ovary, muscle, eye, liver, spleen, heart, and alimentary canal in the rainbow trout, Oncorhynchus mykiss, was examined. All the organs/tissues examined synthesized the 12-lipoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), and 12-hydroxyeicosapentaenoic acid (12-HEPE), implying the widespread nature of this enzyme in trout. Both prostaglandin E and LTC were also found in variable amounts in the organs, with the greatest amount of PGE found in the gill. Leukotriene (LT) B4 and LTB5 were found in supernatants from calcium ionophore-challenged brain, skin, ovary, liver, spleen, and heart, but the lipoxins A4 and A5 were only present in brain, ovary, and spleen in relatively small amounts. As lipoxins have previously been shown to be synthesized by macrophages in rainbow trout [Pettitt et al., J. Biol. Chem. 266, 8720-8726 (1991)], and related cells (microglial cells) are found in the brain of mammals, the localization of macrophage-like cells in trout brain was investigated immunocytochemically. Monoclonal antibodies specific for trout leucocytes failed to identify any microglial-like cells in sections of the brain, although microvessels containing immuno-positive reaction products were observed. A number of distinct lipoxygenase products were found in supernatants of ionophore-challenged gill, including 14-hydroxydocosahexaenoic acid, 12-HETE, and 12-HEPE, and a large number of dihydroxy fatty acid derivatives with conjugated triene chromophores. One of these products was tentatively identified as 8(R),15(S)-dihydroxyeicosatetraenoic acid, a dual 12- and 15-lipoxygenase product, but apparently no LTB4 was generated by this tissue.

Eicosanoid generation and effects on the aggregation of thrombocytes from the rainbow trout, Oncorhynchus mykiss.

Fish blood lacks anucleate platelets but contains a nucleated cell type termed the thrombocyte that is thought to be functionally analogous. Thrombocytes were purified from the peripheral blood of the rainbow trout, Oncorhynchus mykiss, by a two step gradient centrifugation method. Following this procedure, the recovered thrombocytes were 78-86% pure as defined by immunoreactivity to a panel of monoclonal antibodies and were of variable morphology from round to spindle-shaped. Incubation of thrombocyte suspensions with either calcium ionophore, A23187, platelet-activating factor or a thromboxane (TX) mimetic, U-46619, generated a range of eicosanoids derived from arachidonic acid including 12-hydroxyeicosatetraenoic acid (12-HETE), TXB2, prostaglandin (PG) E2, leukotriene (LT) B4 and lipoxin (LX) A4. The equivalent products derived from eicosapentaenoic acid were also formed. Co-incubation of thrombocytes with either erythrocytes or granulocytes/monocytes in the presence of calcium ionophore did not result in the formation of any further new lipoxygenase products. Incubation of isolated thrombocytes in plasma-free conditions with U-46619 (0.03-10 microM) resulted in a rapid, dose-dependent aggregatory response. This effect was markedly augmented in the presence of mammalian fibrinogen (400 micrograms ml-1). Thrombin (0.1-1.3 units ml-1), like U-46619, was also a potent proaggregatory compound for trout thrombocytes. LXA4 and LTB4 had limited aggregatory potential and then only at high concentrations (10 microM), while 12-HETE and PAD had no significant effect at all concentrations tested. These results demonstrate that some of the eicosanoids released during the activation of trout thrombocytes are involved in the aggregatory behaviour of this cell type.