Biological effects of tumor necrosis factor a (TNF-a) are mediated through two cell surface receptors, the 55-kDa TNF receptor and the 75-kDa TNF receptor. The present study investigated the relative roles of the two TNF receptors in normal hematopoiesis. Using agonists (antibodies) specific for the 55-and 75-kDa TNF receptors, we demonstrate differential roles of the two TNF receptors in hemat es in that only the 55-kDa TNF receptor mediates antiproliferative effects ofTNF-aon mature Lin-hematopoietic progenitor cells responding to granulocyte colony-stimulating factor or interleukin 3 alone. In contrast, the 75-kDa TNF receptor is essentil in mediating inhibition of primitive Lin-Sca-l+ highproliferative-potential colony-forming cells and inhibition of the total number of proliferative clones ofindividually cultured Lin-Sca-1+Rh123A and Lin-Sca-1+Rh123hi cells.Tumor necrosis factor a (TNF-a) is recognized as a pleiotropic cytokine (1) with pronounced effects on hematopoiesis (2-6). We have previously demonstrated that the direct effects of TNF-a on mature murine bone marrow progenitors are only inhibitory, whereas stimulatory effects were indirect through induction of cytokine production (6). In this regard, TNF-a is known as a potent inducer of cytokine production (7-9). Although other recent studies have suggested that TNF-a can potentiate early hematopoiesis (10, 11), these studies have not used highly enriched populations of primitive progenitors. Thus, the direct effects on TNF-a on primitive hematopoietic progenitor cells in vitro have not yet been established.In humans and mice, two TNF receptors (TNF-Rs) have been identified and cloned with molecular masses of 55 kDa (TNF-R1) and 75 kDa (TNF-R2) (12-16). The two TNF-Rs show no homology between their intracellular domains, suggesting that they utilize separate signaling pathways (17). Although most cell types appear to express both TNF-R1 and TNF-R2 (13-16), TNF-R1 exclusively mediates most activities of , whereas the known activities mediated through TNF-R2 are restricted to proliferation of T cells (21). The relative roles of the two TNF-Rs in regulation of hematopoietic progenitor cell growth have not been established but are of uttermost interest since TNF mutants with selective activity for either TNF-R1 or TNF-R2 are explored for potential therapeutic applications and may prove to have less adverse effects than TNF-a (22, 23).MATERIALS AND METHODS Growth Factors. Purified recombinant human (rh) granulocyte colony-stimulating factor (G-CSF) and recombinant murine (rm) granulocyte/macrophage colony-stimulating factor (GM-CSF) were generously supplied by Ian K. McNiece (Amgen Biologicals); rm interleukin (IL)-3 was purchased from Promega; rh macrophage colony-stimulating factor (CSF-1) was generously supplied by Michael Geier (Cetus); rmTNF-a was kindly supplied by Genentech; rhTNF-a was a gift from Werner Lesslauer (Hoffmann-La Roche); and rhIL-1a was kindly supplied by Alvin Stern and Peter T. Lomedico (Hoffmann-La Roche). Unless otherwi...

Stem cell factor (SCF), a key regulator of hematopoiesis, potently synergizes with a number of hematopoietic growth factors. However, little is known about growth factors capable of inhibiting the actions of SCF. TNF-a has been shown to act as a bidirectional regulator of myeloid cell proliferation and differentiation. This study was designed to examine interactions between TNF-a and SCF. Here, we demonstrate that TNF-a potently and directly inhibits SCF-stimulated proliferation of CD34 + hematopoietic progenitor cells. Furthermore, TNF-a blocked all colony formation stimulated by SCF in combination with granulocyte colony-stimulating factor (CSF) or CSF-1. The synergistic effect of SCF observed in combination with GM-CSF or IL-3 was also inhibited by TNF-a, resulting in colony numbers similar to those obtained in the absence of SCF. These effects of TNFa were mediated through the p55 TNF receptor, whereas little or no inhibition was signaled through the p75 TNF receptor. Finally, TNF-a downregulated c-kit cell-surface expression on CD34+ bone marrow cells, and this was predominantly a p55 TNF receptor-mediated event as well. (J.

Tumor necrosis factor alpha (TNF alpha) has previously been reported to have both inhibitory and stimulatory effects on hematopoietic progenitor cells. Specifically, TNF alpha has been proposed to stimulate early hematopoiesis in humans. In the present study we show that TNF alpha, in a dose-dependent fashion, can potently inhibit the growth of primitive high proliferative potential colony-forming cells (HPP-CFCs) stimulated by multiple cytokine combinations. Using agonistic antibodies to the p55 and p75 TNF receptors or TNF alpha mutants specific for either of the two TNF receptors, we show that both receptors can mediate this inhibition. In contrast, the potent stimulation of interleukin-3 (IL-3) plus granulocyte-macrophage colony- stimulating factor (GM-CSF) induced HPP-CFC colony formation observed at low concentrations of TNF alpha (2 ng/mL) was only a p55-mediated event. Moreover, the stimulatory effects of TNF alpha on GM-CSF or IL-3- induced colony formation, as well as the inhibition of G-CSF-induced colony growth, were also exclusively signaled through the p55 TNF receptor. Taken together, our results suggest that the inhibitory effects of TNF alpha on primitive bone marrow progenitor cells are mediated through both p55 and p75 TNF receptors, whereas the p55 receptor exclusively mediates the bidirectional effects on more mature, single factor-responsive bone marrow progenitor cells as well as stimulation of IL-3 plus GM-CSF-induced HPP-CFC colony growth.

Tumor necrosis factor-alpha (TNF-alpha) is a bifunctional regulator of hematopoiesis, and its cellular responses are mediated by two distinct cell surface receptors. TNF-alpha generally inhibits the growth of primitive murine hematopoietic progenitor cells (Lin-Scal+) in response to multiple cytokine combinations, and the p75 TNF receptor is essential in signaling such inhibition. In the present study we show the reverse phenomenon in that TNF-alpha on the same progenitor cell population in combination with stem cell factor (SCF) and interleukin-7 (IL-7) through the p55 TNF receptor can recruit additional progenitors to proliferate. In contrast, TGF-beta 1, another bifunctional regulator of hematopoietic progenitor cell growth, completely blocked SCF plus IL- 7-induced proliferation. TNF-alpha increased the number of responding progenitors, as well as the size of the colonies formed. The synergistic effects of TNF-alpha were seen at the single cell level, suggesting that its effects are directly mediated. Finally, whereas SCF plus IL-7 promoted primarily granulopoiesis, the addition of TNF-alpha switched the differentiation toward the production of almost exclusively macrophages.

Snnlmc*ryInterleukin 7 (IL-7) has been demonstrated to be an important regulator of the growth of B and T cell precursors as well as mature T cells, whereas IL-7 has been reported to have no direct myeloproliferative effects. Here we show that IL-7 potently and directly enhances colony stimulating factor-induced myeloid colony formation from Lin-Sca-1 § murine bone marrow progenitor cells, increasing the cloning frequency up to ninefold and cell numbers up to 50-fold, without affecting their ability to differentiate along the myeloid lineages In contrast, IL-7 has no effect on proliferation of committed Lin-myeloid progenitors. Thus, in addition to its established lymphopoietic potential, this study implicates a novel role of IL-7 in early myelopoiesis.