Tumor Necrosis Factor a Directly and Indirectly Regulates Hematopoietic Progenitor Cell Proliferation: Role of Colony-stimulating Factor Receptor Modulation By Sten E. W. Jacobsen,* Francis W. Ruscetti,* Claire M. Dubois,* and Jonathan R. Keller*
From the *Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, and ~Biological Carcinogenesisand Development Program, Program Resources, lnc/D]mCortx, National Cancer Institute-Freden'ck Cancer Researck and Development Center, Frederick, Mar~land 21702
Summary Tumor necrosis factor a (TNF-ce) has been shown to both stimulate and inhibit the proliferation of hematopoietic progenitor cells (HPCs) in vitro, but its mechanisms of action are not known. We demonstrate that the direct effects of TNF-o~ on murine bone marrow progenitors are only inhibitory and mediated at least in part through downmodulation of colony-stimulating factor receptor (CSF-R) expression. The stimulatory effects of TNF-c~ are indirectly mediated through production of hematopoietic growth factors, which subsequently results in increased granulo- cyte-macrophage CSF and interleukin 3 receptor expression. In addition, the effects of TNF-c~ (stimulatory or inhibitory) are strictly dependent on the particular CSF stimulating growth as well as the concentration of TNF-ot present in culture. A model is proposed to explain how TNF-ce might directly and indirectly regulate HPC growth through modulation of CSF-R expression.
he survival, proliferation, and differentiation of hema- TNF-c~ is mainly a monocyte-derived cytokine (16), but T topoietic progenitor cells (HPCs) t are controlled by its production can also be induced in other cell types such positive and negative regulatory cytokines. Stimulatory he- as T cells (17) and endothelial cells (18). TNF-c~ was origi- matopoietic growth factors (HGFs) include the CSFs (1, 2), nally characterized by its cytotoxic and cytostatic activity on the interleukins (3), erythropoietin (4), and stem cell factor tumor cells in vitro and in vivo (19, 20). However, TNF-ot (SCF) (5, 6). The CSFs include granulocyte CSF (G-CSF), has subsequently been demonstrated to affect a wide range macrophage CSF (M-CSF or CSF-1), granulocyte-macrophage of biological activities of many normal cell types, including CSF (GM-CSF), and I1.-3 (2, 7). Presence of one or more endothelium (21), fibroblasts (22), T cells (23), neutrophils of the CSFs is essential for the in vitro proliferation of bone (24), and monocytes (25). Conflicting results have been marrow progenitor cells (2, 7). Biological actions of the CSFs reported with regard to the effects of TNF-ot on the growth are mediated through specifichigh-affanity cell surface receptors and proliferation of HPCs, from potent inhibition (14, 15, (2), and modulation of CSF-R expression has been proposed 26-30) to stimulation (31-33). Furthermore, TNF-cr can pro- as a mechanism by which non-CSF HGFs can dicit inhibi- mote hematopoietic recovery from lethal radiation damage tory and/or stimulatory effects on CSF-stimulated hemato- (34-36). It is not known whether the effects of TNF-c~ on poiesis (8, 9). HPCs are direct and/or indirect, or by which mechanism(s) In addition, a number of negative regulators of hemato- TNF-ot exerts its effects. However, TNF-oe has been demon- poietic cell growth have been identified such as TGF-fl (10), strated to induce the production of a variety of HGFs, in- macrophage inflammatory protein-1 c~ (11), interferons (12), cluding IL-1, IIr M-CSF, G-CSF, and GM-CSF (37-42), prostaglandins (13), and TNF-c~ (14, 15). and to modulate CSF-R expression on granulocytes, macro- phages, and leukemic cells (43-47). Therefore, we addressed whether the stimulatory and inhibitory effects of TNF-cr on hematopoietic cell growth were direct or indirect, and whether 'Abbreviations used in this paper: CM, conditioned medium; HGFs, CSF-tL modulation might represent a mechanism of TNF-(x hematopoietic growth factors; HPCs, hematopoietic progenitor cells; Hu, human; LDBM, light density bone marrow; Lin-, lineage negative; Mu, action. The present results demonstrate that TNF-cr directly murine; PDGF, platelet-derived growth factor; SCF, stem cell factor. inhibits CSF-stimulated proliferation of murine bone marrow
1759 The Journal of Experimental Medicine Volume 175 June 1992 1759-1772 progenitor cells, which directly correlates with CSF-R down- mingen (San Diego, CA). Then, anti-rat Ig-HTC (Cappel Labora- modulation. In addition, TNF-ot has potent stimuhtory effects tories, Malvern, PA) were added at 1:20 dilution and incubated for that are indirect and mediated, at least in part, through produc- 30 min at 4"C, and Thy-l+ cells were separated by FACS| (Becton tion of HGFs and subsequent induction of CSF-Rs. Dickinson & Co.) as previously described (49). Cell Lines Materials and Methods The 32DC13 progenitor cell line (50) was maintained in com- plete RPMI 1640 supplemented with 20% WEHI-3 conditioned Growth Factors medium. The NPS/N1.M6 (MC-6) mast cell line (51), a generous Purified rMuGM-CSF and rHuman (rHuG-CSF) were gener- gift from Dr. Doughs E. Williams (Immunex Corp., Seattle, WA), ously supplied by Ian K. McNiece and Thomas Boone (Amgen was grown in complete R.PMI 1640 with 20% WEHI-3 condi- Corporation, Thousand Oaks, CA). Recombinant routine (rMu) tioned medium. IL-3 was purchased from R and D Systems (Minneapolis, MN). rHuCSF-1 was a generous gift from Dr. MichaelGeier (Cetus Corp., Soft Agar Colony Formation Emeryville, CA). rMuTNF-ot was a gift from Mike Palladino (Genentech, San Francisco, CA). A modification of the method of Stanley et al. (52) was used to measure colony formation of murine bone marrow progenitor Cytokine Antibodies cells in vitro. Briefly, 5 x 104 LDBM cells or 104 Lin- cells in 1 ml of complete IMDM and 0.3% seaplaque agarose were plated Rabbit anti-routine TNF-cr a polyvalent antiserum, was pur- in 35-ram Lux petri dishes (Miles Scientific, Naperville, IL) and chased from Genzyme Corp. (Boston, MA). It had a neutralizing incubated at 37~ in 5% COz for 7 d and scored for colony activity of 10~ U/m1. Goat anti-murine Ib6 was from IL and D growth (>50 cells). Systems, and, depending on the assay, 2-3/~g/ml of this antibody [~H]Thymidine Incorporation Assays. LDBM cells were in- neutralizes 1 ng/ml of routine Ib6. Goat anti-routine IL-1/3 was cubated in the presence of CSFs at 37~ 5% CO2 or TNF- purchased from R and D Systems. Rabbit anti-mouse IlAo~ poly- a-conditioned medium (CM) in 96-well microtiter plates at a den- clonal antibody (Genzyme Corp.) neutralizes 10 U of routine IL-lc~ sity of 5 x 104 cells in 100/~1 of complete IMDM. DNA syn- at a dilution of 1:50. Lyophilizedrat anti-murine CSF-1 mAb was thesis was assessed with a pulse of 1/~Ci of [3H]thymidine (6.7 purchased from Oncogene Science (Manhasset, NY). Ci/mmol; New England Nuclear, Boston, MA) for the last 6 h Preparation of CM. Light density bone marrow (LDBM) cells of the incubation period. Radioactivity was determined by liquid were incubated at 5 x 10s cells/ml in complete IMDM at 370C, scintillation. 5% CO2 in the absence or presence of 2 ng/ml of rMdrNF-c~. Single-CellProliferation Assay. Lin- or Lin-Thy-l+ were seeded Cell-free superaatants were harvested after a 24-h incubation. To in Terasaki plates (Nunc, Kamstrup, Denmark) at a concentration neutralize the effects of TNF-o~ in the CM, it was pretreated with of 1 cell per well in 20/A of complete IMDM. Wells were scored a TNF-o~ antibody (Genzyme Corp.) at a concentration sufficient for proliferation (>10 cells) after 6-8-d incubation at 37~ 5% to neutralize 10 ng/ml of TNF-o, CO2.
Bone Marrow Cells Radioiodination of CSFs Normal murine bone marrow cells were obtained by aspirating The radioiodination of IL-3 was performed by a chloramine-T femurs of normal BALB/c mice. LDBM cells were separated by method as previouslydescribed (53), while G-CSF, CSF-1, and GM- centtifugationon lymphocyteseparation medium (Organon Teknika CSF were labeled by a modifiedchloramine-T method. Briefly,5-10 Corp., Durham, NC). Cells were washed twice in IMDM and /zg ofrMuGM-CSF, recombinant human (rHu)G-CSF, or rHuCSF- resuspended in IMDM supplemented with 10% FCS (Inovar, I in 10/zl of 0.1 tool/liter sodium phosphate buffer (pH 7.0), 10 Gaithersburg, MD), 15 rag/liter gentamicin, and 3 rag/m1 ghta- p.l of 10% DMSO with 100/~g//zl of polyethylene glycol, and 1 mine (complete IMDM). mCi of 1~I (Amersham, Arlington Heights, IL) were incubated at 4*C for 5 rain in the presence of 10/11 of 0.1 mg/ral chlora- Purification of Lin- Bone Marrow Progenitors mine-T. Then, 10/~l of 0.3 mg/ml sodium metabisulfite and 10 Lin- bone marrow progenitor cells were purified according to /zl of 0.1 tool/liter potassium iodide was added. Iodinated CSFs a previously described protocol (48). Briefly, LDBM cells were were separated on a Sephadex G-10 or G-25 column equilibrated resuspended in complete IMDM and incubated at 4oc for 30 rain with PBS. The specific radioactivity was 7.2-10.2 x 106, 0.9-2.0 with a cocktail of antibodies, RA3-6B2 (B220 antigen) and R.B6- x l& 1.7-2.8 x 10% and 6.4-7.8 x 106 cpm/pmol for radio- 8C5 (GR-1 antigen) (gifts of R. Coffman, DNAX Corp., Palo iodinated GM-CSF, G-CSF, IL-3, and CSF-1, respectively.The bi- Alto, CA); MAC-1 (purchasedfrom Boehringer-Mannheim, Indi- ological activity of all CSFs were retained for at least 3--4 wk after anapolis, IN); Lyt-2 (CD8) and L3T4 (CD4) (purchasedfrom Becton radiolabeling, as determined by their ability to induce bone marrow Dickinson & Co., Sunnyvale, CA). Cells were washed twice and proliferation. resuspended in complete IMDM. Magnetic beads (Dynal, Great RadioligandBinning Experirnentl [ml]CSFbinding experiments Neck, NY) were added at a ratio of 40:1 (beads/cells), and the mix- were performed by a previously described phthalate oil separation ture was incubated for 30 min at 4~ Labeled (Lin+) cells were method (54). LDBM cells or Lin- cells were resuspended in 1 ml removed by a magnetic particle concentrator (Dynal), and Lin- of 50 mM glycin~HCl (PH 3.0) for 1 re_into releasebound ligands. cells were recovered from the supernatant. Cells were then washed twice in RPMI 1640 containing 1% BSA, Purificationand FACS Sorting of Lin-Thy-1 + Progenitors. Lin- 20 retool/liter Hepes, and 0.1% sodium azide (binding medium). cells were indirectly labeled with the mAbs anti-Thy-l.2 (Becton Cells were incubated with 1~I-cytokines in 200/~l of binding Dickinson) or an isotype-matched control purchased from Phar- medium. Spedfic binding of radiolabeled CSFs (20-300 pM) was
1760 TNF: Mechanisms of Action in Hematopoiesis determined as the difference in CSF binding in the absence and and a goat anti-rat-FI~2 (Cappel Laboratories) as previously de- presence of a 50-fold excess of unlabeled CSF following a 90-rain scribed (9). Cells were analyzed by FACS~ on a EPIC-753 (Coulter incubation at 22~ (GM-CSF) or 37~ (G-CSF, CSF-1, and ID3). Electronics, Hialeah, FL). Cell-bound [12slJCSF was separated from unbound ligands by cen- trifugation through a 0.2-ml mixture of dibutylphthalate and 1 Results isis (2 ethyl-hexyl) phthahte oil (ratio, 1.5:1) (Eastman Kodak, Roch- ester, NY). Radioactivity was measured in a Biogamma 2 counter Effects of TNF~ on CSlZinduced Clonal Growth of Murine (Beckman Instruments, Fullerton, CA). Equilibrium binding data Bone Marrow Progenitor Cells In Vitra Previous studies had were analyzed according to Scatchard (55) and by computerized demonstrated considerable variability in the effects of TNF-c~ linear regression analysis. on the growth of HPCs in vitro, ranging from potent inhi- bition to stimulation (14, 15, 26-33). Because many of these Cell Surface Phenotyping studies used CM as a source of colony stimulating activity LDBM cells were labeled with and-LFA-1, anti-MAC-I, and (CSA) as well as unfractionated bone marrow, we first as- anti-Ly-5 (Boehringer-Mannheim), anti-Ly-17 (Dr. Margareth L sessed the effects of rMuTNF-ce over a broad dose range on Hibbs, Melbourne, Australh), or RB6-8C5 (gift from Dr. Coffman), the growth of CFU cells (CFU-C) induced by purified rCSFs.
A G-CSF I~'/~ CSF-1 200 GM-CSF II]]]]]l IL-3 ._T_.
150
C 0 0 20 200 100 0.02 0.2 2.0 1 0.02 0.2 2.0 20 200 0.02 0.2 2.0 20 200 0.02 0.2 2.0 20 200 r ! i
U. o I 50 I I -f_
TN F-c<, nglml
B 200 m
Z_ Figure 1. Effectsof TNF-c, on CSF- 150 - stimulated colony formation of LDBM m cells and Lin- progenitors.LDBM (A) r and Lin- cells (B), separated as de- O O scribed in Materials and Methods, were "6 plated at 5 x 104 and 2 x 104 cells, 20 200 respectively, in 1 ml of IMDM with 100 0.02 0.2 2.0 0.02 0,2 2,0 20 200 0.02 0.2 2.0 20 200 0.02 0.2 2.0 20 200 10% FCS and 0.35% seaphque agaros~ =i Cultures were supplementedwith pre- O determined optimal concentrations of I purified rMuGM-CSF (20 ng/ml), I.I. r rHuG-CSF (20 ng/ml), rMull.-3 (20 50- ng/ml), or rHuCSF-1 (50 ng/ml), and incubated at 37~ 5% CO2 for 7 d before being scored for colony forma- tion (>50 cells). Results are presented as the mean +SD of duplicate deter- minations and are representativeof at 0 - TNF-cq nglml least four separate experiments.
1761 Jacobsen et al. In addition, we compared the effects of TNF-ol on normal ng/ml, respectively.The same response profileto TNF-ol was LDBM cells versus a highly enriched bone marrow progen- observed on CSF-l-stimulated Lin- progenitors (Fig. 1 B). itor cell population, designated lineage negative (Lin-) due Similar to the results with CSF-I, IL-3-stimulated colony to its absence of cell surface antigens characteristic of B and formation was enhanced at low concentrations of TNF-cr T lymphocytes, monocytes, and granulocytes (48). (0.02-2.0 ng/ml), with a maximum of 43% and 33% for Although TNF-o~ alone did not promote colony forma- LDBM (Fig. 1, A) and Lin- cells (Fig. 1, B), respectively, tion of LDBM cells (data not shown), it had a bidirectional whereas 20-200 ng/ml had less or no stimulatory effect. effect on G-CSr'-induced colony formation (Fig. 1 A). Finally, and in contrast to the colony formation induced Specifically, concentrations of TNF-c~ at 2 ng/ml or below by the other three CSFs, GM-CSF-induced CFU-C in soft enhanced the number of G-CSF--stimulated CFU-C, with agar was increased at all concentrations of TNF-o~ tested a 44% increase at 0.02 ng/ml and maximum enhancement (0.02-200 ng/ml). Maximum enhancement of 83% for of 75% at 2 ng/ml. In contrast, TNF-cr at 20 and 200 ng/ml LDBM (Fig. 1 A) and 74% for Lin- cells (Fig. 1 B) was inhibited G-CSF-stimulated colony formation by 72% and observed at 20 ng/ml. Furthermore, the TNF-cc-induced en- 83%, respectively. A similar bidirectional response to TNF-c~ hancement of GM-CSF-stimulated colony formation was was observed on Lin- progenitors, in that G-CSr,-stimu- specific and not due to contaminants such as LPS, since an lated CFU-C formation was maximally enhanced by 58% antibody to TNF-ol neutralized its stirnulatory activity (data at 2 ng/ml, whereas a 70% and 80% inhibition was observed not shown). at 20 and 200 ng/ml, respectively (Fig. 1 B). Thus, TNF-o~ has bidirectional effects on routine bone TNF-cz also stimulated CSP-l-induced CFU-C formation marrow colony formation induced by purified rCSFs that is of LDBM cells (Fig. 1 A). TNF-a increased the number of dependent on the particular CSF stimulating growth as well CSF-l-induced CFU-C by 37% at 0.02 ng/ml, and a max- as the concentration of TNF-ol in culture. imum stimulation of 92% was observed at 2 ng/ml of TNr,-cx. TNF-cr Is a Bidirectional Modulator of CSF-R Expression on In contrast to the effects observed on G-CSF-induced colony HPCs. BecauseTNF-ot had been shown to downmodulate growth, higher concentrations of TNF-o~ did not inhibit CSF- G-CSF and GM-CSF-Rs on human granulocytes (44, 45) and 1-induced colony formation, with a marginal increase in CSF-11k expression on routine monocytes (43, 47), we ex- colony formation of 28% and 17% observed at 20 and 200 amined whether modulation of CSF-R expression could play
A ~ 3000 - E 25001I o 2500 .t _T.. 2000 O 2000m _T_ @ : 1500 ~ 1500 o o
O 1000 -- 1000 ? ff 500 ? 5oo
15" 1 h 6h 12h 24h 15" 1 h 6h 12h 24 h TNF-ce Incubation Time TNF-e Incubation Time
E E 2500 o 5000 T I o Figure 2. The effects of TNF-(x on 400C .t 2000 CSF binding to LDBM cells. LDBM cells (5 x 105 cells/ml) were incubated u _T_ at 37~ in the presence of TNF-c~ 20 3000 o 1500 o ng/ml. 12SI-G-CSF(A), t2sI-CSF'-I(B), t2SI-GM-CSF (C), and t2sI-ID3 (D) 2000 1ooo specific binding were determined fol- lowing various incubation periods as tie- 1000 500 scribed in Materials and Methods. i Results are presented as the mean • of duplicate determinations and are rep- 0 15" 1 h 6 h 12 h :4 h 0 15" 1 h 6h 12h 24 h resentative of at least four separate ex- TNF-a Incubation Time Incubation Time perirnents.
1762 TNF: Mechanisms of Action in Hematopoiesis a role in the bidirectional effects of TNF-c~ on bone marrow and II:3 binding of 91% and 47%, respectively, was observed. progenitor cell proliferation. TNF-ol (20 ng/ml) reduced This upregulation was prolonged for at least 48 h (data not lzSI-G-CSF-specific binding to LDBM cells in a time- shown). Furthermore, the specific binding of all four CSFs, dependent manner (Fig. 2 A). Maximum downmodulation were not significantly affected after a 24-h incubation in con- of G-CSF binding of 86% was observed by 15 min, which trol medium (data not shown). was sustained for at least 24 h (Fig. 2 A) and was reversed Equilibrium binding studies and subsequent Scatchard anal- after removal of TNF-c~ (data not shown). CSF-1 binding ysis revealed that untreated LDBM cells had 253 high-affinity was maximally reduced 87% by 15 min, remained suppressed (KD -- 1.1 x 10 -l~ M) G-CSF binding sites per cell and for 12 h, but unlike G-CSF binding, recovered to 73% of that TNF-c~ at 20 ng/ml by 1 h reduced the number of control binding by 24 h (Fig. 2 B). GM-CSF (Fig. 2 C) and G-CSF-Rs to 80 without significantly affecting the receptor Ib3 binding (Fig. 2 D) were also rapidly downmodulated affinity (Fig. 3 A). Similarly, TNF-o~ 20 ng/ml reduced the by TNF-c~, with a maximum reduction by 1 h of 54% and number of both high- and Iow-afflnity GM-CSF-Rs by 51% 52%, respectively. However, in contrast to G-CSF and CSF-1 and 53%, respectively, after 1 h without affecting their affini- binding, both GM-CSF and II-3 binding recovered to con- ties (Fig. 3 B). trol levels within 12 h, and by 24 h an increase in GM-CSF TNF-cz-induced modulation of CSF-Rs occurred in the
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0.02 Figure 3. Scatchard analysis of TNF 1763 Jacobsen et al. Table 1. Modulation of CSF Binding to Lin- Bone Marrow in LDBM, the effect of TNF-ce on CSF-R expression was Progenitor Cells by TNF-oz also investigated on Lin- bone marrow progenitor cells (Table 1). Their high proliferative potential is underscored by the Specific binding finding that up to one in five Lin- cells proliferate under Incubation optimal conditions in single-cell cultures supplemented with Ligand time - TNF-oe + TNF-oe multiple HGFs (Keller, J. R., S. E. '07. Jacobsen, and F. W. Kuscetti, unpublished observations). As observed for LDBM h cpm cells, TNF-ce reduced the binding of all four CSFs to Lin- G-CSF 1 494 + 42 85 • 23* ceils by 1 h and upregulated GM-CSF and II.-3R expression G-CSF 24 549 + 78 123 + 59* by 187% and 64%, respectively, at 24 h (Table 1). CSF-1 1 2,348 • 213 389 + 671 To address the specifidty of TNF-ce-induced modulation CSF-1 24 2,052 • 341 1,363 + 134" of CSF-Rs, the expression of other ceil surface antigens on GM-CSF 1 1,980 + 102 1,073 + 48" LDBM ceils detected by mAb were examined by FACS"~ anal- ysis after TNF-ot treatment. TNF-ce (20 ng/ml) did not affect GM-CSF 24 1,867 • 159 3,491 • 341" the expression of LFA-1, MAC-l, Ly-5, Ly-17, or 8C5 by 1 h IL-3 1 1,468 _+ 84 809 • 91* (Table 2) or 6 h (data not shown). IL-3 24 1,281 • 71 2,101 • 130~ Differential Regulation of G-CSF-R Expression Depends on TNF-oz Concentration. Because TNF-o~ optimally down- Lin- bone marrow cells were separated as described in Materials and modulated the expression of all four CSF-Rs by 1 h, we chose Methods, and incubated at 5 x 105 cells/ml at 37~ 5% CO2 in the to investigate the concentration-response relationship at this presence or absence of TNF-oe 20 ng/ml. CSF-specific binding was de- time point. TNF-ce-induced downmodulation of G-CSF- and termined after a 1- and 24-h incubation as described in Materials and CSF-l-specific binding (Fig. 4), as well as GM-CSF- and IL- Methods. Data are presented as the mean of duplicate determinatiom + SD 3-specific binding (data not shown), occurred in a concen- and are representative of at least three separate experiments. Results of three experiments were analyzed by t tests comparing -TNF-ot vs. tration-dependent manner, with maximum inhibition at 20 + TNF-a. ng/ml and an ED50 of 0.2-2.0 ng/ml. "p .~0.01. In seeming conflict, TNF-ce downmoduhted G-CSF specific * F 4g0.05. binding at low concentrations (0.2 ng/ml) by 1 h (Fig. 3), whereas G-CSF-stimulated colony formation was enhanced absence of changes in cell numbers and viability (data not at the same concentration (Fig. 1). However, because colony shown), and was not due to TNF-ce binding to the CSF-Rs, formation occurs only after several days of incubation (56), because TNF-ce did not compete for CSF binding sites at we examined whether high concentrations of TNF-oe (20-200 4~ (data not shown). ng/ml), which inhibit colony formation, might correhte with Because bone marrow progenitor cells are in low frequency prolonged downmodulation of G-CSF binding, whereas low 3000 Table 2. Effect of TNF-oz on the Expression of Cell Su~ace Proteins on LDBM Cells Percent positive cells Mean ~uorescence E ~" 2000 .=_ mAb - TNF-tx + TNF-tx - TNF-o~ + TNF-ot ~= o Control <5 <5 4 3 LFA-t 46 41 7 8 u.~ 1000 MAC-1 34 30 29 33 0 Ly-5 82 79 43 37 Ly-17 17 21 4 4 8C5 47 53 43 42 0 0.02I 0.2t 2.01 210 200I TNF-u, nglrnl LDBM cells were incubated at 5 x 105 ceUs/ml at 37~ 5% CO2 for 1 h in the presence or absence of TNF-ot 20 ng/ml. Cells were then in- Figure 4. Dose-response of TNF-cc-induced downmodulation of G-CSF cubated with primary mAbs and a secondary fluorescein conjugated Ab and CSF-1 binding to LDBM cells. LDBM cells (5 x 105 cells/ml) were as described in Materials and Methods. An isotype-matched rat Ig was incubated at 37~ in the presence of increasing concentrations of TNF-ce used as a control. Percent positive cells refers to the percentage of cells or in the absence of growth factors, lzsI-G-CSF (@) and 12sI-CSF-1 (A) with a higher fluorescence intensity than 95% of the cells incubated with specific binding was determined as described in Materials and Methods the control Ab. Results presented are representative for three separate after a 1-h incubation. Results are presented as the mean of triplicate de- experiments. terminations _+SD and are representative of three separate experiments. 1764 TNF: Mechanisms of Action in Hematopoiesis Table 3. Relaaonship between Dose- and Time-Dependence of Medium TNF-ol-induced Downmodulation of G.CSF i---'] G-CSF ~'.'.~ CSF- 1 Binding on LDBM Cells GM-CSF o [rErrrl IL-3 I~I-G-CSF specific binding x 6 oE c TNF-c~ 15 min 24 h o ng/ral cpra 0 2,384 + 182 2,104 + 167 2 I 0.2 1,597 _+ 152" 2,013 _+ 87 I 2.0 691 _+ 54* 1,906 _+ 192 I 20 481 _+ 41" 452 _+ 103" I 200 439 + 79* 513 + 110" -- + - + TNF-~ LDBM cells were incubated at 5 x 10s cells/ml at 37~ 5% CO2 in 16 the absence or presence of TNF-ot at indicated concentrations. G-CSF- specific binding was determined as described in Materials and Methods o after 15-rain and 24-h incubation. Data presented are the mean of dupli- x 12 cate determinations _+ SD and are representative of at least three separate E experiments. Results of three experiments were analyzed by analysis of o variance followed by t tests comparing -TNF-~x vs. + TNF-ot. * p g0.01. gs c concentrations (0.2-2 ng/ml), which enhance colony forma- tion, might correlate with recovery of G-CSF binding. In- i" terestingly, TNF-cz reduced G-CSF specificbinding to LDBM ceUs by 33% at 0.2 ng/ml and by 71% at 2.0 ng/ml after 15 min, whereas no significant effect was observed by 24 h + at the same concentrations of TNF 1765 Jacobsen et al. Table 4. Direct Effects of TNF~ on CSF-dependent Growth and 1:21 for IL-3 (Table 4). TNF-ot at 2.0 ng/ml, a concen- of Separated Bone Marrow Progenitor Populations tration that enhanced colony formation in response to all four CSFs (Fig. 1), reduced the frequency of responding single Growth in cells to 1:600 for G-CSF, 1:60 for CSF-1, 1:39 for GM-CSF, Terasaki plates and 1:32 for IL,3-treated cultures, respectively (Table 4). Similar degrees of inhibition were also observed adding TNF-ot at Percent 20 ng/ml (data not shown). TNF-ot was also examined for Cells CSF TNF-ot Frequency-1 Inhibition direct effects on a very primitive progenitor cell population within the Lin- cells, characterized by cell surface expres- Lin - -- -- 0 -- sion of the Thy-1 antigen (48). The proliferation of single Lin - -- + 0 -- Thy-l+/Lin- cells supplemented with CSF-1, GM-CSF, or Lin - G-CSF - 75 - IL-3 were also inhibited by TNF-c~ at 2.0 ng/ml (Table 4) Lin - G-CSF + 600 88" and 20 ng/ml (data not shown). Lin - CSF-1 - 33 - Thus, in striking contrast to the stimulatory effects ob- served on colony formation in soft agar, TNF-c~ directly in- Lin - CSF-1 + 60 45* hibits G-CSF-, CSF-I-, GM-CSF-, and IL-3-induced prolifer- Lin - GM-CSF - 22 - ation of murine bone marrow progenitor cells in single cell Lin - GM-CSF + 39 44* assays. Lin - IL-3 - 21 - Indirect Stimulator), Effects of TNF-cr : Proposed Mechanism Lin - IL-3 + 32 34* of Action. Because TNF-ot acted as a direct inhibitor of single bone marrow progenitors, it was possible that the stimula- Lin-Thy-1 + G-CSF - 0 0 tory effects of TNF-c~ on CSF-induced colony formation as Lin-Thy-1 + G-CSF + 0 0 well as increased GM-CSF and Ib3K expression were indirectly Lin-Thy-1 + CSF-1 - 75 - mediated through induction of other cytokines. TNF-ot has Lin-Thy-1 + CSF-1 + 125 40 t been demonstrated to induce the production of a number Lin-Thy-1 + GM-CSF - 30 - cytokines, including the CSFs, II--1, and Ib6 (37-42). Fur- Lin-Thy-1 + GM-CSF + 50 40* thermore, synergistic effects on the growth of bone marrow Lin-Thy-1 + IL-3 - 8 - progenitor cells have been shown to occur between different CSFs (58, 59) as well as between CSFs and IL-1 and IL-6 (8, Lin-Thy-1 + IL-3 + 15 47$ 60, 61). Therefore, we first examined the CM of bone marrow cells exposed to TNF-ot 2 ng/ml for 24 h (TNF-o~-CM) for Lin - and Lin-Thy-1 + cells separated according to Materials and Methods synergistic activity on GM-CSF-induced proliferation as well were seeded as single cells in Terasaki plates as described. Cultures were as GM-CSF-K expression on LDBM progenitor cells. TNF-ol incubated at 37~ 5% CO2 with predetermined optimal concentrations at 2 ng/ml stimulated GM-CSF-induced proliferation of of CSFs as described in Fig. 1, in the presence (+) or absence (-) of 2 ng/ml of TNF-ot. The frequency of responding progenitors (>10 LDBM cells by 57% after 48 h (Table 5). In comparison, cells/well) was determined after a 6-7-d incubation. Results presented TNF-o~-CM enhanced GM-CSF-stimulated proliferation of are the mean of four separate experiments, with a minimum of 1,200 LDBM cells by 39%, and this enhancement was also seen wells scored per group. Results of three experiments were analyzed by in the presence of a TNF-a antibody (Table 5). In addition, paired t tests comparing -TNF-c~ vs. + TNF-o~. TNF-ot-CM upregulated GM-CSF-spedfic binding after 24 h "p ~0.01. p ~0.05. both in the presence (59%) and absence (70%) of anti- TNF-c~ (Table 5). Upregulation of the GM-CSF-K expres- sion was also observed at 0.2 ng/ml and 20 ng/ml of TNF-ot enhanced by 78%, 54%, and 35%, respectively (Fig. 5 C). (data not shown). In contrast, the non-TNF-ot-treated con- Thus, transient downmodulation of CSF-Ks (GM-CSF-K, trol CM had no such activity (Table 5). Thus, the stimula- CSF-I-R, and IL-3-K) correlates with a transient inhibition tory effects of TNF-ot on GM-CSF-induced proliferation and of thymidine uptake, whereas prolonged downmodulation on GM-CSF-K expression of murine bone marrow cells are (G-CSF-K) is accompanied by sustained inlfibition. Conversely, indirect. TNF-ot-induced upregulation of GM-CSF and Ib3Ks (by To determine whether the indirect effects of TNF-ot were 24 h) and recovery of CSF-1Ks correlated with enhanced mediated through induction of other cytokines, mAbs against proliferative responsiveness. murine CSF-1, Ib3, Iblol, Iblfl, and IL-6 were examined TNF-cr Directly Inhibits the Proliferation of Murine Bone Marrow for neutralizing activity on colony formation stimulated by Progenitors in Response to CSFs. Because indirect effects cannot the combination of TNF-ol and GM-CSF (Table 6). The CSF-1 be ruled out in soft agar colony assays, direct proliferative antibody significantly (25-30%) inhibited the TNF-o~ stimula- effects of TNF-ot were assessed by examining the growth of tory effect on GM-CSF-induced CFU-C, whereas the other single Lin- ceils in Terasaki plates. In agreement with previ- antibodies had no significant effect alone (Table 6) or in com- ously published data (57), the responding frequencies of Lin- bination (data not shown). ceils were 1:75 for G-CSF, 1:33 for CSF-1, 1:22 for GM-CSF, Because G-CSF has been demonstrated to possess potent 1766 TNF: Mechanisms of Action in Hematopoiesis Table 5. Effect of CM of TNF-cr-treated Bone Marrow Cells on GM-CSF-induced Proliferation and GM-CSF-R Expression on LDBM Progenitors [3H]TdR lzSI-GM-CSF- Stimulator* Anti-TNF-oet incorporations specific binding" cpm clam GM-CSF - 16,540 _+ 1,781 2,352 +_ 145 GM-CSF + 15,265 _+ 1,524 2,384 _+ 173 GM-CSF + TNF-o~ - 25,927 + 2,4171 4,825 _+ 2871 GM-CSF + TNF-oe + 15,627 _+ 1,739 2,194 _+ 149 GM-CSF + control CM - 18,920 -+ 1,491 2,219 + 87 GM-CSF + control CM + 16,793 _+ 1,034 2,109 -+ 107 GM-CSF + TNF-c~-CM - 22,994 -+ 2,1391 3,994 _+ 2131 GM-CSF + TNF-cz-CM + 23,739 -+ 1,8051 3,512 _+ 1691 * LDBM cells were incubated in 2 ng/ml of GM-CSF, 2 ng/ml of TNF-~, or in 50% CM from TNF-oe-treated or -untreated (control) LDBM cells, prepared as described in Materials and Methods. * The cultures were supplemented with a rabbit anti-mouse TNF-oe antiserum ( + ) or a control serum ( - ) as described in Materials and Methods. $ [3H]TdR incorporation was determined as described in Materials and Methods, after a 48-h incubation at 37~ 5% CO2 in the presence of 2 ng/ml of GM-CSF. Results are the mean of triplicate determinations • SD and representative of three separate experiments. II lzSI-GM-CSF-specific binding to 2.0 x 106 LDBM cells was determined as described in Materials and Methods after a 24-h incubation. Data are the mean of duplicate determinations • SD and representative of three experiments. Results of this experiment were analyzed by t test using a pooled estimate of error. <0.01. Table 6. Neutralizing Effects of Hematopoietic Growth Factor synergistic effects on CSFol-, GM-CSF-, and IL-3-induced Antibodies on TNF-c~-induced Enhancement of GM-CSF-stimulated colony formation (58) and because a mAb against murine Colony Formation G-CSF was not available, we examined the ability of TNF- oe-CM to stimulate the proliferation of the 32DC13 cell line. TNF-ol Antibody* Colony formation (CFU-C) As previously demonstrated, this cell line proliferates in re- sponse to II.-3 and G-CSF (50), but not to other hematopoi- - - 45_+4 + - 81 _+ 5* 14 + TNF-c~ 48 _+ 7* - CSF-1 42 -+ 3 12 x + CSF-1 69 -+ 3 g lO - IL-3 40 _+ 7 c 0 + IL-3 87 _+ 9 = 8 - IL-6 39 _+ 5 + IL-6 76 _+ 6 ~ 4 - IL-lc~ 49 _+ 6 D,- + IL-lc~ 73 _+ 8 2 - IL-I~ 51 _+ 2 + IL-1/3 92 _+ 10 // 1024.... 512 256 128 ~'4 3'2 1'8 8' 4' 2' CM Dilution -~ Lin - bone marrow cells were plated at 2 x 104 cells in 1 ml of com- Figure 6. G-CSF activity in CM from TNF-oe-stimulated LDBM cells. plete IMDM and 0.35% seaplaque agarose, supplemented with 20 ng/ml The 32DG13 cell line (@) and NFS/N1.M6 cell line (A) were incubated of rMuGM-CSF and incubated at 37~ 5% CO2 in the absence (-) at 104 cells/well at 37~ in the presence of increasing concentrations of or presence of 2 ng/ml of TNF-a. Colony growth (>50 cells) was scored CM from TNF-~v-stimulated LDBM cells (prepared as described in Materials after a 7-d incubation. and Methods). [3H]TdR incorporation was determined as described in * Cultures were supplemented with optimal concentrations of anti-mouse Materials and Methods after a 24-h incubation. Data presented are the cyrokine antibodies described in Materials and Methods before addition mean of triplicate determinations • and are representative of three ex- of TNF-ot. Results presented are the mean of duplicate determinations periments. 32DC13 cells stimulated by 20 ng/ml of G-CSF and 20 ng/ml • SD and are representative of three separate experiments. Results of this of IL-3 incorporated 40,609 • 5,737 and 47,717 • 6,198 cpm, respec- experiment were analyzed by t tests using a pooled estimate of error. tively, whereas SCF- and IL-3-stimulated MC-6 cells incorporated 7,900 p <0.01. • 738 and 39,867 • 3,524 cpm, respectively. 1767 Jacobsen et al. etic growth factors such as SCF, CSF-1, GM-CSF, II.-1, Ib2, Discussion Ib4, Ib5, Ib6, and II.-7 (Keller, J. R., unpublished observa- .Although initial studies suggested that the effects of TNF-ce tions). TNF-c~-CM stimulated the [3H]TdR incorporation on bone marrow colony growth in ~tro were only inhibi- of 32DC13 cells in a dose-dependent manner in the presence tory (14, 15, 26-33), more recent studies have proposed a of a TNF-ol antibody (Fig. 6). stimulatory role for TNF-o~ in hematopoiesis (19-21). In the The recently identified SCF is also a potent synergistic factor present study, we investigated the reason for this apparent (5, 6), and because a SCF antibody was not available, the discrepancy. CM of TNF-c~-stimulated LDBM cells was treated for SCF The data presented here confirm that the TNF-c< is a bi- activity on the NFS/N1.M6 (MC-6) mast cell line. This cell directional modulator of HPC proliferation, but demonstrate line proliferates in response to Ib3, Ib4, and SCF but not that direct effects of TNF-cx on murine bone marrow cells G-CSF (51). The TNF-ol-CM did not promote the growth and highly enriched multipotential progenitors are only in- of MC-6 cells, indicating that the supernatants did not con- hibitory. These inhibitory effects of TNF-c~ were correlated tain Ib3, IL-4, or SCF (Fig. 6). with its ability to trans-downmodulate the receptor expres- G-CSF but Not CSF-1 Mimics TNF-cr-induced Upregulation sion for the CSFs stimulating growth. In addition, TNF-o~ of GM-CSF and IL.3R Expression. To investigate whether indirectly stimulates CSF-stimulated bone marrow colony for- TNF-c~-induced upregulation of GM-CSF and IL-3R expres- mation through at least the induction of G-CSF and CSF-1. sion could be mediated by CSF production, Lin- progen- Some TNF-o~-inducible cytokines, such as G-CSF, might itor cells were incubated in the presence or absence of 20 ng/ml mediate the observed synergistic response through upregula- of G-CSF or 50 rig/m1 of CSF-1, and examined for GM-CSF tion of CSF-R expression. Fig. 7 is a schematic presentation and IL-3 binding after 24 h. G-CSF increased GM-CSF- and of how these pathways might interact to mediate the Ib3-specific binding by 137% and 49%, respectively (Table pleiotropic effects of TNF-c~. 7), without significantly affecting cell number or viability TNF-c~ rapidly reduced the expression of all four CSF-Rs (data not shown). In contrast, CSF-1 did not significantly affect GM-CSF- or Ib3-specific binding to Lin- cells (Table 7). Furthermore, in agreement with the inability of TNF-o~ G-CSF receptor Time to increase CSF-1R expression (Fig. 2), G-CSF did not GM-CSF and 0 signiticantly affect CSF-1R expression by 24 h (Table 7). IL-3 receptors Taken together, these data indicated that the stimulatory effects of TNF-c~ on GM-CSF- and IL-3-induced bone marrow progenitor cell proliferation are indirect and mediated, through ~ TNF-a HGF production, and subsequent upregulation of GM-CSF High or Low and II.-3R expression. 15 min Table 7. G.CSF but Not CSF-I Upregulates GM-CSF and IL-3R Expression on Lin- Progenitor Cells ~zsI-CSF-spedfic binding G'C'S',o .others ~. r A Stimulator GM-CSF IL-3 CSF-1 24 h c~m Medium 2,135 + 132 1,678 -+ 89 2,397 _+ 219 G-CSF 5,060 + 268" 2,501 + 192" 2,693 _+ 184 1 1 CSF-1 2,468 ,+ 214 1,892 _+ 173 1,838 _+ 149" Responsiveness ~' G-CSF 4' IL-3 + GM-CSF ~' Lin- cells (5 x 10s cells/ml) were incubated at 37~ 5% CO2 for 24 h in the presence of 20 ng/ml of G-CSF or 50 ng/ml of CSF-1, or in the absence of growth factors. 12SI-GM-CSF-, tzsI-IL-3-, and 12sI- Figure 7. TNF-o 1768 TNF: Mechanisms of Action in Hematopoiesis on LDBM cells as well as Lin- progenitor cells. This cor- GM-CSF-Rs than to elicit proliferation of hematopoietic pro- related to reduced responsiveness to all four CSFs in the short- genitor cells. term [3H]TdR incorporation assay, as well as inhibition in The use of Lin- bone marrow cells was important to dem- the single-cell cloning experiments. The magnitude of inhi- onstrate that modulation of CSF-R expression occurred also bition was closely correlated to the magnitude of receptor on enriched progenitor cells. Lin- sdection of bone marrow downregnhtion. Furthermore, the downmodnlation of CSF-Rs progenitors removes T and B cells and macrophages; how- is specific since TNF-ot neither affected the expression of a ever, no selection for bone marrow stromal cells such as fibro- number of other cell surface proteins nor the expression of blasts and endothdium was used. It was therefore not unex- c-kit, another growth factor receptor on HPCs (Dubois, pected that TNF-ot indirectly enhanced colony formation of C. M., F. W. Ruscetti, andJ. R. Keller, manuscript in prep- Lin- cells nearly as much as for LDBM cells, whereas they aration). were directly inhibited in single cell assays. Adherence deple- Previous studies demonstrated that TNF-ot induced a tran- tion can reduce but not eliminate these indirect effects sient reduction in the expression of CSF-1Rs on murine mac- (Jacobsen, S. E. W., unpublished observations). These findings rophages (43, 47). Because this correlated with synergistic underscore that negative selection of HPCs does not neces- growth stimulation, it was proposed that TNF-ot might mimic sarily reduce the extent of indirect effects and that the ques- the biological action of CSF-1 by downmodulating CSF-1Rs. tion of indirect effects of biological modifiers on such pro- However, in those studies, [3H]TdP-, incorporation was mea- genitors can only be properly addressed by the use of single-cell sured only after a 48-h incubation, at which time CSF-1R cloning experiments. Although it is not possible to deter- expression had returned to initial or even greater levels (43, mine receptor expression on single cells, the present data sug- 47). Here, we dearly demonstrate that the initial downmodu- gest that TNF-~x could directly induce prolonged downmodu- lation of CSF-1Rs results in a transient inhibition of lation of all CSF-Rs. [3H]TdR incorporation. In addition, the effects of TNF-ot It was possible that the TNF-ot-induced downmodulation on CSF-l-stimulated single bone marrow progenitor cells of CSF-Rs observed in the present study was mediated through are only inhibitory. Additional support for the concept that induction of cytokines capable of reducing CSF-R expres- CSF-1R trans-downmodulation results in reduced rather than sion. However, we have not been able to detect such activity enhanced CSF-1 responsiveness comes from a study demon- in the supernatants after a 15-60-min incubation with TNF-ot strating that I/.-3- and GM-CSF-induced downmodulation (S. E. W. Jacobsen, unpublished observations). It also seems of CSF-1K expression on a myeloid precursor cell line sup- unlikely that TNF-cz-induced cell death or changes in prolifer- presses the biological responsiveness to CSF-1 (62). ation could mediate the reduction in CSF binding. This is In contrast to its direct inhibitory effects, the delayed based on the observation that TNF-ot-induced downmodu- stimulatory effects of TNF-ot on CSF-I-, GM-CSF-, and lation of CSF-Rs occurred within minutes and that TNF-ol IL-3-induced proliferation are indirect and mediated at least alone did not affect the proliferation or viability of bone partially through production of CSF-1 and G-CSF, as detected marrow progenitors as compared to untreated control cells, by neutralizing antibodies and the ability of TNF-cc-CM to even after a 24-h incubation. induce proliferation of the G-CSF responsive 32DC13 cell It has recently been demonstrated that TNF-cz can stimu- line. This conclusion is also supported by previous work late the proliferation and upregulate the expression of GM- showing that TNF-ot is an inducer of G-CSF production (38) CSF and Ib3Rs on human acute myeloid leukemia cells (46). and that potent synergistic interactions occur between G-CSF The mechanism of action was not investigated; however, the and the three other CSFs (58, 63, 64). It was further sub- present data suggest that the induction of other cytokines stantiated by the ability of G-CSF to mimic the delayed could mediate the observed GM-CSF and Ib3R upregula- TNF-ot-induced upregnlation of GM-CSF and IL.3R expres- tion and enhanced responsiveness to these cytokines. In sup- sion. Furthermore, G-CSF did not affect CSF-1R levels port of such an indirect mechanism, another study demon- significantly, suggesting that the synergistic activity of G-CSF strated that the synergistic activity of TNF-ot on GM-CSF- on IL-3- and GM-CSF-, but not on CSF-l-stimulated colony stimulated AML proliferation could be partially neutralized formation could partially be mediated through receptor in- by an antibody to Ibl (65). duction. In further support of such a mechanism, we found Bidirectional dose-dependent proliferative effects are not that the increase in GM-CSF and IL-3R expression preceded unique to TNF-ot. In parallel to what we have observed for the synergistic activity observed on [3H]TdR incorporation. TNF-ot, Battegay et al. (66) showed that TGF-~ induces au- Because TNF-c~ downmodulated G-CSF-K expression by tocrine platelet-derived growth factor (PDGF) secretion of 86%, it could be argued that the low level of G-CSF-Rs might connective tissue cells at low concentrations, whereas it down- be insu~cient to mediate the G-CSF--induced upregulation modulates the ot-subunits of the PDGF-R at higher concen- of GM-CSF-Rs proposed in the present model. However, we trations. The physiological relevance of such bidirectional have recently found that exogenously added G-CSF (20 ng/ml) concentration-dependent effects on HPCs are hard to assess can upregulate GM-CSF-R expression in the presence of high since the local concentrations of these modulators in the bone concentration of TNF-ct (20 ng/ml) (Jacobsen, S. E. W., et marrow microenvironment are unknown. al., unpublished observations). This suggests that lower levels The biological significance of growth factor receptor modu- of G-CSF-R occupancy are required to induce expression of lation as a mechanism for cell growth regulation has been 1769 Jacobsen et al. disputed, because it has been shown for hematopoietic (53, average of only 100-300 of each CSF-R (2). In the present 67) as well as nonhematopoietic cells (68) that only a frac- study, TNF=c~ reduced the number of G=CSF-Rs from 304 tion of growth factor receptors need their ligand bound to to 96, which could put many progenitors below threshold elicit a maximum proliferative response (the spare receptor receptor level needed to induce a proliferative response. concept). A more recent study did however show that as many Although it seems likely that also other pathways mediate as 50% of the CSF=ILs must be occupied to induce optimal the pleiotropic actions of TNF=ot in hematopoiesis (16), the proliferation of hematopoietic progenitors (69). In addition, data presented here implicate a role of CSF=R modulation the spare receptor concept was established on cell lines with in the direct inhibitory as well as the indirect stimulatory high levels of growth factor receptors, whereas bone marrow effects of TNF=ot on murine bone marrow progenitor cells progenitors display profound heterogeneity and express an (summarized in Fig. 7). We thank Drs. Dan L. Longo and Joost J. Oppenheim for helpful discussions and review of this manu- script, and also Charles W. Riggs for statistical analysis of the data. This research was sponsored by the National Cancer Institute, Department of Health and Human Set= vices, under contract no. NO1=CO=74102 with Program Resources, Inc./DynCorp. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 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