Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenito r cell proliferation in vitro, and cytokine combinations that include SF ca n support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primiti ve status while proliferating. These cells also display higher cell-specifi c cytokine uptake rates than more differentiated types of hematopoietic cel ls. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetic s of SF receptor (c-kit) internalization by human Mo7e cells exposed to dif ferent extracellular concentrations of soluble SF. Transfer of Mo7e cells t o a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellu lar SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the c ells continued to deplete SF from the medium but at a much slower rate. Dur ing this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-ma ss transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvi ronment of the cells. Taken together, these results underscore the importan ce of monitoring and appropriately regulating cytokine concentrations in he matopoietic cell expansion cultures. They may also help to explain the diff erent biological responses exhibited by primitive hematopoietic cells expos ed to different types and concentrations of cytokines for periods of days. (C) 1999 John Wiley & Sons, Inc.