Recombinant human erythropoietin induces proliferation and Ca(2+)-influx in specific leukocyte subpopulations of rainbow trout (Oncorhynchus mykiss) blood and head kidney cells

Abstract

In mammals, erythropoietin regulates the development and differentiation of erythrocytes. Although hematopoietic cells of bony fish correspond in their ontogeneic development, morphology, and function to their mammalian counterparts, an erythropoietin (EPO)-like molecule has not been identified. In this study we present evidence for a mitogenic response of blood and head kidney leukocytes of rainbow trout after stimulation by recombinant human EPO (rhu EPO). The modulation of cellular activities is accompanied by the induction of DNA-binding activities in nuclear extracts of these cells. In addition, flow cytometric analysis of intracellular Ca2+ concentrations revealed a long-lasting and rhu EPO dose-dependent increase, which was shown to be abrogated by cross-aggregation of surface IgM using anti-trout-IgM monoclonal antibodies (mabs). In flow cytometric dual-labeling experiments using rhu EPO/anti-EPO antiserum and mabs specific for trout leukocyte subpopulations, it was shown that a subpopulation of trout B-cells binds rhu EPO. Moreover, in a modified Ca2+ activation assay, it was demonstrated that this blood B-cell subpopulation is the rhu EPO responder population. In conclusion, the data suggest the existence of EPO-binding receptors in trout that are able to trigger Ca(2+)-independent intracellular signaling in hematopoietic cells of head kidney and Ca(2+)-dependent activation of a subpopulation of B-lymphocytes.