Tumor necrosis factor responsiveness of hematopoietic stem cells in mouse long-term bone marrow cultures

Abstract

In vitro studies of cytokine effects on isolated hematopoietic stem cells have yielded conflicting results. Tumor necrosis factor-alpha (TNF) is one such cytokine that has been shown to have both positive and negative effects. We used the long-term bone marrow culture (LTBMC) system of Dexter as a model to study the role of TNF in the regulation of hematopoiesis. In this model, differentiation of stem cells into committed precursors occurs. Mycophenolic acid (MPA) treatment of LTBMC eliminates hematopoietic progenitors and leaves an intact stromal cell hematopoietic microenvironment. The addition of populations enriched for stem cells (stromally depleted) to these MPA purified stromal microenvironments allows for the study of stem cell repopulating kinetics and growth factor responsiveness as they interact with their natural microenvironment. We have found that TNF has pleiotropic effects on a wide range of progenitors. A primitive progenitor (day 12 CFU-S) is positively regulated in the cultures, while, at the same time, another primitive progenitor (HPP-CFC), as well as a more mature progenitor (CFU-C) are negatively regulated. In order to determine whether TNF causes these effects by itself or induces other cytokines, RNA was extracted and supernatants harvested from TNF-treated and control cultures. TNF induced increases in CSF-1, TNF and IL-1. Because fresh MPA treated stromal layers contain no colony forming activity in either soft agar or in day 12 CFU-S assays, TNF was added to MPA-treated cultures not recharged with fresh marrow to determine the effect of TNF on purified stroma. TNF caused an immediate burst of hematopoiesis, with the first evidence of hematopoiesis appearing at day 2 post-MPA treatment followed by the appearance of assayable progenitors by day 7. Control MPA-treated cultures showed no signs of hematopoiesis. This indicates that TNF stimulates a very primitive and quiescent progenitor spared by MPA treatment. However, of the cytokines found to be induced by TNF, only IL-1 was able to stimulate the MPA-resistant long-term culture initiating cell (LTC-IC). These experiments provide evidence that TNF regulates both non-cycling hematopoietic stem cells as well as committed progenitors in long-term cultures.