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Two tumor necrosis factor receptors (TNFRs) with molecular weights of 55 kD (TNFR-p55) and 75 kD (TNFR-p75) have recently been identified and cloned. In previous studies, TNFR-p55 has been shown to exclusively mediate bidirectional effects of TNF-alpha on committed bone marrow granulocyte-macrophage progenitor cells, whereas both TNFR-p55 and TNFR- p75 can mediate inhibition of primitive progenitors requiring multiple cytokines to proliferate. We show here that TNF-alpha potently and directly inhibits the in vitro growth of committed erythroid progenitor cells in response to multiple cytokine combinations, and that TNF-alpha- induced inhibition of burst-forming unit-erythroid colony formation is mainly mediated through TNFR-p55, although TNFR-p75-mediated inhibition could be observed on progenitors responsive to erythropoietin alone. Moreover, at low TNF-alpha concentrations (2 ng/mL), TNF-alpha stimulates interleukin-3-dependent in vitro growth of committed granulocyte-macrophage progenitor cells, whereas it potently inhibits erythroid progenitor cell proliferation, showing that one concentration of TNF-alpha can simultaneously and bidirectionally modulate interleukin-3-dependent growth of committed granulocyte-macrophage (stimulation) and erythroid progenitor cells (inhibition).

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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.