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The Journal of Immunology, 2005, 175: 1464-1472.
Copyright © 2005 by The American Association of Immunologists

Multiple Members of the TNF Superfamily Contribute to IFN-{gamma}-Mediated Inhibition of Erythropoiesis1

Nadia Felli2,*, Francesca Pedini2,*, Ann Zeuner*,{dagger}, Eleonora Petrucci*, Ugo Testa*, Concetta Conticello{dagger}, Mauro Biffoni*, Andrea Di Cataldo{ddagger}, Jeffrey A. Winkles§, Cesare Peschle* and Ruggero De Maria3,*,{dagger}

* Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy; {dagger} IOM Foundation, Catania, Italy; {ddagger} Pediatric Hematology and Oncology Unit, University of Catania, Italy; and § Departments of Surgery and Physiology, University of Maryland School of Medicine, Baltimore, MD


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma} inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-{gamma} up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-{gamma} in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-{gamma} on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-{gamma} inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-{gamma}-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-{gamma}-mediated regulation of hematopoiesis and show that the effects of IFN-{gamma} on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemopoietic stem cells give rise to mature erythroid cells through a series of intermediate differentiation stages including the burst-forming unit-erythroid, colony-forming unit-erythroid (CFU-E), and erythroid precursor cells. The maturation of erythroid precursors occurs in peculiar cellular associations named erythroblastic islands, which consist of a central macrophage surrounded by maturing erythroid precursors. In the final step of erythroblast maturation, terminally differentiated orthochromatic erythroblasts extrude the nucleus and leave the bone marrow as circulating reticulocytes (1). This gradual wave of diffeentiation/maturation is positively regulated by erythropoietin (Epo).4 The binding of Epo to its specific cell surface receptor, expressed on both CFU-E and erythroid precursor cells, increases cell proliferation and progression through erythroid maturation and induces the expression of antiapoptotic genes, such as Bcl-2 and Bcl-XL (2, 3, 4).

Effective erythroid maturation depends upon intracellular signaling networks that regulate cell growth and apoptosis. Recent studies have provided evidence that signals produced by TNF family members and specifically by death receptors (DR) play a central role in erythroid homeostasis both in physiological and pathological conditions (5, 6, 7, 8, 9). Immature erythroid precursors at the prebasophilic/basophilic stage are susceptible to DR stimulation by TNF family ligands such as CD95 ligand (CD95L) or TRAIL (5, 7). DR cross-linking on immature erythroblasts induces caspase-mediated degradation of the key erythroid transcription factors SCL and GATA-1, resulting either in erythroblast death or in a reversible inhibition of erythropoiesis depending on the levels of available Epo (6, 10). Therefore, the maintenance of RBC homeostasis ultimately depends on the finely regulated balance between positive signals elicited by stimulatory cytokines such as Epo and c-kit ligand and negative signals delivered by death receptor ligands and inhibitory cytokines such as IFN-{gamma} (11, 12, 13). IFN-{gamma} has been shown to inhibit the growth and differentiation of erythroid precursor cells and has been proposed as a potential mediator of hemopoietic failure in aplastic anemia (14, 15, 16). IFN-{gamma} treatment of differentiating erythroid progenitors is known to increase CD95 expression, which contributes to the suppression of cell growth and differentiation (14). However, in that interfering with CD95 function results in an incomplete restoration of proliferation and colony-forming activity of IFN-{gamma}-treated erythroblasts (14), it is likely that other factors besides CD95 activation arbitrate IFN-{gamma}-mediated suppression of erythropoiesis.

TWEAK (Apo-3 ligand) is a new member of the TNF family with almost ubiquitous mRNA expression (17, 18, 19). TWEAK is synthesized as a type II membrane protein, although in vitro studies suggest that it can also be secreted by producer cells (17). The result of target cell stimulation by membrane or soluble TWEAK largely depends on the cellular context. In vascular endothelial cells, TWEAK induces proliferation, migration, and angiogenesis (20, 21, 22, 23), whereas TWEAK expressed by human peripheral blood monocytes upon IFN-{gamma} stimulation has been shown to be involved in cytotoxicity against tumor cells (24).

Whereas early studies reported that TWEAK could induce cell death through binding of WSL-1/TRAMP (DR3) (19), other reports have questioned this finding and showed that TWEAK was able to transduce signals in cells lacking DR3 expression (25, 26). DR3-independent signaling has been ascribed to a recently identified receptor for TWEAK, originally cloned as a mitogen-inducible gene in murine fibroblasts and named fibroblast growth factor-inducible 14 (Fn14) (27, 28). Fn14 is distantly related to other members of the TNFR family and contains one cysteine-rich domain in the extracellular region and a TNFR-associated factor-binding domain in the cytoplasmic region (29, 30). Despite its small size and the absence of a cytoplasmic death domain, Fn14 has been recently shown to mediate multiple pathways of TWEAK-induced cell death in transformed cells, including both caspase-dependent apoptosis and cathepsin B-dependent necrosis (31).

In this study, we show that treatment of purified human erythroblasts with IFN-{gamma} results in inhibition of growth and differentiation and modulates the expression of several TNF/TNFR family members including TWEAK, TRAIL, and Fn14. TWEAK and its receptor Fn14 were expressed at low levels during the differentiation of human erythroid progenitors and were able to negatively modulate the proliferation and differentiation of erythroid precursor cells. Only the combined neutralization of CD95L, TWEAK, and TRAIL was able to restore erythroid cell survival, proliferation, and maturation in the presence of IFN-{gamma}, indicating the simultaneous involvement of TWEAK, TRAIL, and CD95L as effectors of IFN-{gamma} in hemopoietic suppression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Growth factors, Abs, and chemicals

Human rIL-3, GM-CSF, G-CSF, M-CSF, IL-6, Flt3 ligand, and IFN-{gamma} were purchased from PeproTech Inc. Human recombinant Epo was provided by Amgen. Soluble human recombinant TWEAK, Fn14:Fc and DR5:Fc were purchased from Alexis Biochemicals. Anti-Human CD95L mAb (clone Nok-1) was obtained from BD Pharmingen. Soluble anti-human Fn14 (clone ITEM-1) Ab was kindly provided by Hideo Yagita (Juntendo University School of Medicine, Tokyo, Japan). zVAD-fmk was purchased from Bachem. Anti-mouse IgG Ab was obtained from Sigma-Aldrich. Human recombinant LZ-TRAIL was kindly provided by Henning Walczak (DKFZ, Heidelberg, Germany) and anti-CD95 agonistic Ab (clone CH11) was purchased from Upstate Biotechnology. The anti-TWEAK Ab (rabbit) was kindly provided by J. P. Medema (Leiden University Medical Center, Leiden, The Netherlands). The anti-Fn14 Ab (rabbit) has been described (28). The anti-{beta}-actin Ab (mouse) was purchased from Oncogene.

Peripheral blood human progenitor cell (HPC) purification and culture

Peripheral blood buffy coat was obtained from healthy donors after their informed consent and approval by the Committee for Human Studies (32). Human CD34+ precursor cells were purified from peripheral blood by positive selection using the midi-MACS immunomagnetic separation system (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of CD34+ cells was determined by flow cytometry using a monoclonal PE-conjugated anti-CD34 Ab (Dako) and was routinely over 96%. CD34+ progenitors were cultured in serum-free medium in the presence of various recombinant human cytokine combinations. Serum-free medium was prepared as follows: IMDM was supplemented with BSA (10 mg/ml), pure human transferrin (0.7 mg/ml), human low-density lipoprotein (40 µg/ml), insulin (10 µg/ml), sodium pyruvate (10–4 M), L-glutamine (2 x 10–3 M), rare inorganic elements supplemented with iron sulfate (4 x 10–8 M), and nucleosides (10 µg/ml each). For erythroid unilineage culture, serum-free medium was supplemented with 0.01 U/ml IL-3, 0.001 ng/ml GM-CSF, and 3 U/ml Epo to induce uncontaminated unilineage erythroid differentiation. In these culture conditions, a progeny of cells 98 ± 2% glycophorin A+ is generated (33).

For monocytic differentiation, CD34+ progenitor cells were grown in liquid suspension cultures supplemented with 40% FBS, IL-6 (1 ng/ml), Flt3-ligand (100 ng/ml) and saturating doses of M-CSF (500 U/ml). Cells were cultured at 37°C in a 5% CO2/5% O2/90% N2 humidified atmosphere. The differentiation stage of unilineage cultures was evaluated by May-Grünwald-Giemsa staining (Sigma-Aldrich) and cytologic analysis. For granulocytic unilineage culture, serum-free medium was supplemented with IL-3 (1 U/ml), GM-CSF (0.1 ng/ml), and saturating amounts of G-CSF (500 U/ml).

For clonogenetic assay, 500 CD34+ were plated in duplicate in 0.9% methylcellulose supplemented with 5% FBS, IL-3 (0.01 U/ml), GM-CSF (0.001 ng/ml), and Epo (3 U/ml) for erythroid differentiation or with 40% FBS, IL-6 (1 ng/ml), Flt3 ligand (100 ng/ml), and M-CSF (500 U/ml) for monocytic differentiation. Colonies were scored after 14 days of culture at 37°C in a 5% CO2/5% O2/90% N2 humidified atmosphere.

Immunofluorescence and flow cytometry analysis

To measure glycophorin A expression, 5 x 105 erythroid cells were washed with cold PBS containing 0.1% sodium azide and incubated for 30 min on ice with optimal amounts of FITC-labeled control IgG or anti-glycophorin A mAbs (BD Pharmingen). Alternatively, to detect transmembrane TWEAK expression, cells were incubated for 30 min on ice with PE-conjugated control IgG or anti-TWEAK (CARL-1) mouse Abs (eBioscience). Cells were washed twice with PBS and relative fluorescence intensity of individual cells was evaluated with a FACScan flow cytometer (BD Biosciences). To detect transmembrane Fn14 expression, cells were incubated for 30 min on ice with control IgG or anti-Fn14 (ITEM-1) mouse Abs. After two washes with PBS, cells were incubated with anti-mouse biotinylated Ab (Molecular Probes), washed again and incubated on ice with PE-conjugated streptavidin (Molecular Probes). For three-color cytofluorimetric analysis, bone marrow mononuclear cells were incubated for 10 min with 6% normal mouse serum and treated with saturating concentrations of directly conjugated anti-glycophorin A FITC and PerCP anti-CD45 (BD Biosciences). Cells were then washed twice in cold PBS/azide. Relative fluorescence intensity of individual cells was evaluated with a FACScan flow cytometer.

Analysis of apoptotic cells

Apoptosis was determined by staining erythroid progenitor cells with Alexa Fluor 488-conjugated annexin V. At day 11 of unilineage culture, 5 x 105 erythroid cells were washed with PBS, resuspended in 100 µl of annexin V binding buffer (10 mM HEPES/140 mM NaCl/2.5 mM CaCl2, pH 7.4) and incubated with 5 µl of annexin V-Alexa Fluor 488 for 15 min at room temperature in the dark. Samples were counterstained with 5 µg/ml propidium iodide, washed with annexin V binding buffer and analyzed with a FACScan flow cytometer.

RT-PCR analysis

Total RNA was extracted by the guanidium isothiocyanate-CsCl method from 1 to 3 x 105 cells in the presence of 12 µg of Escherichia coli rRNA. RNA samples (200 ng to 1 µg) were reverse-transcribed (Moloney murine leukemia virus-RT; Invitrogen Life Technologies) with oligo-(dT) (Invitrogen Life Technologies) as primers. Complementary DNA amounts were equalized by RT-PCR using primers for the S26 gene. Amplification of ~5 ng of RT-RNA within the linear range was achieved with 23 PCR cycles (i.e., this cycle number allowed a linear RT-RNA dose response). The amplification procedure involved denaturation at 95°C for 45 s, annealing for 45 s, extension at 72°C for 45 s. The primers used are listed below: S26, forward 5'-GCCTCCAAGATGACAAAG-3', reverse 5'-CCAGAGAATAGCCTGTCT-3' (56°C); TWEAK, forward 5'-CCAGATCGGGGAGTTTATAG-3', reverse 5'-GAAGGGGGCAGCCTTGAGAT-3' (58°C); Fn14, forward 5'-CCTCACGCTGGCTCACAC-3', reverse 5'-GGGGGGGTCTGTATCTTA-3' (56°C); TL1A, forward 5'-AGTTCCAGGCTCTAAAAG-3', reverse 5'-TCTGGCTTGTTTGGTCGG-3' (52°C); DR3, forward 5'-CGCAGGTGACATGGTCCT-3', reverse 5'-CTTGAGCATCTCGTACTG-3' (57°C); TRAIL, forward 5'-AACCTCTGAGGAAACCAT-3', reverse 5'-TTAGCCAACTAAAAAGGC-3' (50°C); TRAIL-R2/DR5, forward 5'-GCCTCATGGACAATGAGATAAAGGTGGCT-3', reverse 5'-CCAAATCTCAAAGTACGCACAAACGG-3' (58°C); TRAIL-R1/DR4, forward 5'-CTGAGCAACGCAGACTCGCTGTCCAC-3', reverse 5'-TCCAAGGACACGGCAGAGCCTGTGCCAT-3' (61°C); TRAIL-R3/DcR1, forward 5'-CGTAGGGATCATAGTTCT-3', reverse 5'-TCACAAGGAGGAAGATAG-3' (52°C); TRAIL-R4/DcR2, forward 5'-CTTTTCCGGCGGCGTTCATGTCCTTC-3', reverse 5'-GTTTCTTCCAGGCTGCTTCCCTTTGTAG-3' (60°C); CD95L, forward 5'-CAAGTCCAACTCAAGGTCCAT-3', reverse 5'-CAGAGAGAGCTCAGATACGTT-3' (55°C); CD95, forward 5'-ACTGTGACCCTTGCACCAA-3', reverse 5'-CTTTCTGTTCTGCTGTGTC-3' (50°C). The PCR was performed in a total volume of 50 µl, using AmpliTaq DNA polymerase (PerkinElmer Life Sciences) according to the manufacturer’s instructions. Following amplification, 10 µl of each sample was separated on a 2% agarose gel and transferred to a nitrocellulose filter (Schleicher and Schuell). Filters were hybridized with specific probes of ~30 bp previously labeled with [{gamma}-32P]ATP using a polynucleotide kinase (Invitrogen Life Technologies). Semiquantitative evaluation of mRNA levels was performed by the ImageQuant software (Molecular Dynamics).

Western blotting

Cell pellets were washed twice with cold PBS and lysed on ice for 30 min with 1% Nonidet P40 lysis buffer (20 mM Tris-HCl, pH 7.2, 200 mM NaCl, 1% Nonidet P40) in the presence of 1 mM PMSF and 2 µg/ml each of aprotinin, leupeptin, and pepstatin. Cell debris was removed by centrifugation at 13,000 rpm for 10 min at 4°C and protein concentration of supernatants was determined using the Bio-Rad protein assay. Aliquots of cell extracts containing 40 µg of total protein was resolved on 12 or 15% SDS-PAGE and transferred to a Hybond-C extra nitrocellulose membrane (Amersham Biosciences).

Filters were blocked for 1 h at room temperature in 5% nonfat dry milk dissolved in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20) and then incubated in 1% BSA/TBST containing a dilution of primary Ab (10 µg/ml anti-TWEAK, 1:500 anti-Fn14, and 1:10,000 anti-{beta}-actin) for 1 h ({beta}-actin) or overnight (TWEAK and Fn14). After washing in TBST buffer, filters were incubated for 45 min in 5% nonfat dry milk dissolved in TBST containing a 1/4,000 dilution of the corresponding peroxidase-conjugated secondary Ab (Amersham Biosciences). Proteins were visualized with the ECL technique (Super Signal West Pico; Pierce) according to the manufacturer’s instructions.

Statistical analysis

The percentage of protection from IFN-{gamma} inhibitory effect was determined as: percentage of protection = [1 – (A1 – A2)/(A1 – A3)] x 100 where A1 is the number of untreated cells, A2 is the number of cells treated with IFN-{gamma} and blocking molecules, A3 is the number of IFN-{gamma}-treated cells. No difference was observed between control cells and cells treated with blocking molecules alone.

The percentage of inhibition exerted by IFN-{gamma}, anti-CD95, TRAIL, and TWEAK stimuli was determined as: percentage of inhibition = 100 – [(B2 x 100)/B1] where B1 is the number of untreated cells, B2 is the number of cells treated with either IFN-{gamma}, TWEAK, LZ-TRAIL, or anti-CD95 Abs.

Paired t test was used to analyze the statistical significance of the experimental results; p values <0.05 were considered significant. Data are presented as mean values ± 1 SD of the mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IFN-{gamma} inhibits the proliferation and differentiation of purified human erythroblasts

IFN-{gamma} is a pleiotropic cytokine secreted by the Th-1 subset of CD4+ lymphocytes and by NK cells, which exerts inhibitory effects on the growth of normal and malignant hemopoietic cells (13, 34). We investigated the effect of IFN-{gamma} on immature human erythroblasts in a liquid culture system that allows the progressive differentiation of virtually pure cell populations belonging to a specific myeloid lineage (33). At day 5 of unilineage differentiation, erythro-myeloid cells are fully committed and proliferate until terminal maturation (33). As expected, IFN-{gamma} exerted a robust inhibition of granulocytic and erythroid cell growth (Fig. 1A). IFN-{gamma} was also able to inhibit the differentiation of early erythroid precursors, as shown by the persistence of basophilic erythroblasts at late stages of culture (Fig. 1B) and by the lower expression of glycophorin A (Fig. 1C). The inhibitory action of IFN-{gamma} on erythropoiesis has been previously attributed to CD95 up-regulation and caspase activation, with subsequent apoptosis of immature erythroid cells (14, 15, 35). However, CD95 neutralization or caspase-3 and -8 inhibition have proved only partially able to block IFN-{gamma}-induced apoptosis and inhibition of erythropoiesis (14, 35). Therefore, we sought to investigate the potential involvement of other members of the TNF family in IFN-{gamma}-mediated hemopoietic suppression.



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  		FIGURE 1. IFN-{gamma} inhibits erythroid cell growth and differentiation. A, IFN-{gamma} inhibits the proliferation of erythroid and granulocytic precursor cells in a dose-dependent manner. Day 5 differentiating erythroid and neutrophilic cells were exposed to increasing amounts of IFN-{gamma}, and counted at day 11 (erythroid) and at day 17 (neutrophilic) of unilineage culture. The results are the mean ± SD of four independent experiments. B and C, May-Grünwald-Giemsa staining (B) and glycophorin A (GpA) expression (C) of cells at day 11 of erythroid culture, untreated or treated with 300 U/ml IFN-{gamma} for 6 days (IFN-{gamma}).

 
IFN-{gamma} up-regulates multiple TNF family members on immature erythroblasts

Developing erythroid cells express CD95 and TRAIL receptors (5, 36). To investigate whether IFN-{gamma} may inhibit erythropoiesis through different effectors, we analyzed the expression of members of the TNF/TNFR family, including TRAIL and the recently characterized proteins TWEAK and TL1A, in IFN-{gamma}-treated erythroid precursor cells. RT-PCR analysis revealed that TWEAK and TRAIL mRNA levels were significantly increased in erythroblasts treated with IFN-{gamma} compared with untreated cells at the same differentiation stage (Fig. 2A), whereas CD95L mRNA expression remained unchanged. The expression of TL1A, the recently characterized ligand for DR3 (37), was undetectable both in unstimulated and IFN-{gamma}-treated erythroblasts, ruling out the possible involvement of DR3-mediated signals in IFN-{gamma}-mediated inhibition of erythropoiesis. IFN-{gamma}-mediated TWEAK and TRAIL up-regulation was confirmed by Western blot analysis on purified erythroblasts at day 7 of differentiation (Fig. 2B). Evaluation of TNFR family receptors after IFN-{gamma} treatment of erythroid precursors confirmed the previously reported increase of CD95 expression and revealed a significant up-regulation of the TWEAK receptor Fn14. TRAIL receptor 2 and TRAIL receptor 4 expression was consistently decreased, whereas TRAIL receptor 3 remained unchanged following treatment with IFN-{gamma} (Fig. 2A). Remarkably, a simultaneous increase in both receptor and ligand expression occurred only in the case of TWEAK/Fn14, suggesting that this receptor-ligand system may play a functional role in IFN-{gamma}-mediated inhibition of erythropoiesis.



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  		FIGURE 2. Expression of TWEAK, TRAIL, CD95L, TL1A, and their receptors in immature erythroblasts untreated or treated with IFN-{gamma}. A, Semiquantitative RT-PCR analysis of erythroblasts at day 6 of differentiation cultured in standard erythroid medium (control) or treated for 24 h with 100 U/ml IFN-{gamma} (IFN-{gamma}). The human erythroleukemic cell line TF-1 was used as positive control for TL1A gene expression. Amplification of the S26 housekeeping gene cDNA (bottom left) was used to normalize RT-RNAs. OD of RT-PCR bands obtained as log ratios between IFN-{gamma}-treated and control cells is shown. The results are the mean ± SD of three different experiments. B, Western blot analysis of TWEAK and TRAIL on day 7 erythroblasts untreated (control) or stimulated for 48 h with 100 U/ml IFN-{gamma} (IFN-{gamma}). OD of immunoblot bands obtained as log ratios between IFN-{gamma}-treated and control cells is shown. The results are the mean ± SD of three different experiments.

 
TWEAK and Fn14 are expressed during erythroid differentiation/maturation

The finding that TWEAK and its receptor are expressed on erythroid precursor cells prompted us to investigate their modulation during erythropoiesis and their functional effect on erythroblast proliferation and differentiation. To evaluate the levels of TWEAK expression during erythropoiesis, we examined TWEAK mRNA levels at discrete stages of erythroid maturation on purified populations of in vitro cultured human erythroblasts. TWEAK mRNA expression could be detected in CD34+ cells and during unilineage erythroid differentiation, with a moderate increase in terminal erythroid precursors (Fig. 3A). Western blot analysis revealed TWEAK expression on erythroid precursors at day 8 of unilineage culture, which was confirmed by flow cytometry (Fig. 3, B and C), but absence of protein expression in CD34+ cells. Fn14 mRNA was present at low levels in CD34+ cells and during all stages of erythroblast differentiation (Fig. 3D). Although almost undetectable in CD34+ cells, Fn14 protein expression in erythroblasts was confirmed by Western blotting and flow cytometry analysis (Fig. 3, E and F).



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  		FIGURE 3. TWEAK and Fn14 expression during unilineage erythroid differentiation. Purified peripheral blood CD34+ cells were cultivated in standard erythroid medium and analyzed for RNA and protein expression. A, Semiquantitative RT-PCR analysis of TWEAK mRNA expression on CD34+ cells and erythroblasts at different days of unilineage culture. The S26 housekeeping gene cDNA (bottom panel) was used to normalize RT-RNAs. A representative result from three independent experiments is shown. B, Western blot analysis of TWEAK expression on CD34+ cells and erythroblasts at day 8 of unilineage culture. The expression of {beta}-actin was used as loading control. The results are shown for cells derived from different donors. C, Flow cytometry analysis of transmembrane TWEAK expression on erythroblasts (E) at day 8 of unilineage differentiation. Cells were stained with anti-TWEAK Ab as described in Materials and Methods. Monocytes (Mo) treated with IFN-{gamma} were used as a positive control; the Burkitt’s lymphoma cell line Raji was used as a negative control. D, Semiquantitative RT-PCR analysis of Fn14 mRNA expression on CD34+ cells and erythroblasts at different days of unilineage culture. The S26 housekeeping gene cDNA (bottom panel) was used to normalize RT-RNAs. A representative experiment of three performed with cells from different donors is shown. E, Western blot analysis of Fn14 on CD34+ cells and erythroblasts at day 8 of differentiation derived from different donors. The HT29 colon adenocarcinoma cell line was used as a positive control, while the expression of {beta}-actin was used as loading control. F, Flow cytometry analysis of surface Fn14 expression on CD45– gated bone marrow (BM) mononuclear cells, and erythroblasts at day 7 (E day 7) and day 14 (E day 14) of unilineage differentiation.

 
TWEAK inhibits erythroid cell growth and differentiation

As TNF family members such as CD95L and TRAIL have been previously reported to be implicated in the negative regulation of erythroid differentiation (5, 6), we hypothesized that TWEAK may exert a similar inhibitory effect on erythropoiesis. Therefore, we treated immature erythroblasts at day 5 of unilineage differentiation with recombinant soluble human TWEAK and assessed cell proliferation and differentiation after 9 additional days of culture. Evaluation of viable cell numbers at day 14 of unilineage culture in the presence of TWEAK revealed a significant inhibition of erythroid cell expansion (Fig. 4A). The inhibitory effect of TWEAK on erythroblast expansion was then compared with that exerted by CD95L and TRAIL, which play a well characterized role in the negative regulation of erythropoiesis. TWEAK displayed a weaker, though significant inhibitory effect on erythroblast growth, while leaving the proliferation of control monocyte cultures unaffected (Fig. 4B). These observations suggest that TWEAK may contribute to the negative regulation of erythropoiesis albeit with lower intensity compared with other death receptor ligands. The analysis of erythroid cell differentiation showed a significant decrease in the proportion of mature (orthochromatic) erythroblasts in TWEAK-treated cultures and a concomitant increase of immature (basophilic and polychromatophilic) erythroblasts (Fig. 4C), thus revealing an inhibitory effect of TWEAK on erythroid maturation. TWEAK did not affect the differentiation of control monocytic cultures (Fig. 4D), suggesting that it acts as a specific regulator of erythroid cell production.



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  		FIGURE 4. TWEAK is an inhibitor of erythroid proliferation and differentiation. A, Day 5 differentiating erythroid and monocytic cells in liquid unilineage system were exposed to different amounts of recombinant soluble TWEAK protein (TWEAK) for 9 days. At day 14, the percentage of growth inhibition was evaluated. B, Day 5 differentiating erythroid and monocytic cells were exposed to 100 ng/ml recombinant soluble TWEAK protein (TWEAK), 100 ng/ml recombinant LZ-TRAIL protein (TRAIL), or 100 ng/ml anti-CD95 Ab (anti-CD95). The percentage of growth inhibition was calculated at day 14 of culture as described in Materials and Methods. The results are the mean ± SD of five independent experiments. C, Representative May-Grünwald-Giemsa stainings (left panels) and percentage of basophilic (Baso) polychromatophilic (Poly) and orthochromatic (Ortho) erythroblasts (right panels) obtained after 14 days of unilineage differentiation in standard erythroid medium (Control) or in the same medium supplemented with 100 ng/ml recombinant soluble TWEAK (TWEAK), starting from day 5 of culture. D, A monocytic culture was used as a control. The percentage of promonocytes (Pro), monocytes (Mo), and macrophages (M{Phi}) was evaluated at day 14. The results are the mean ± SD of five independent experiments. *p < 0.05 and ***p < 0.0001 vs control.

 
Fn14 stimulation impairs the clonogenetic activity of erythroid progenitors via caspase activation

To confirm that Fn14 triggering impairs erythroid cell growth and differentiation, we investigated the ability of an agonistic anti-Fn14 Ab to reduce erythroid cell formation and maturation in liquid and semisolid cultures. Day 5 erythroid cells were exposed to 5 µg/ml anti-Fn14 (ITEM-1) in the presence or absence of the pan-caspase inhibitor zVAD. Evaluation of cell number showed that ITEM-1 significantly reduced the number of erythroid cells generated in liquid cultures (Fig. 5A). The anti-erythropoietic effects of TWEAK and Fn14 were further evaluated in clonogenetic assays, which showed reduced colony formation, size, and hemoglobinization in semisolid cultures treated with either TWEAK or ITEM-1 (Fig. 5, B and C). Thus, Fn14 stimulation reduces erythroid cell growth and differentiation by acting at single cell level. Although TWEAK may transduce death signals in tumor cells by both caspase activation and formation of reactive oxygen intermediates (31), the caspase inhibitor zVAD was able to completely prevent the anti-proliferative effect of ITEM-1 (Fig. 5A), indicating that caspases mediate the negative regulation of erythropoiesis induced by TWEAK.



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  		FIGURE 5. Fn14 stimulation impairs the clonogenetic activity of erythroid progenitors via caspase activation. A, Purified peripheral blood CD34+ cells undergoing unilineage erythroid differentiation were treated from day 5 with mouse IgG or 5 µg/ml anti-Fn14 (ITEM-1) in the presence or absence of the pan-caspase inhibitor zVAD (40 µM). Erythroid cell growth was evaluated until day 15 of differentiation. The results are the mean ± SD of three independent experiments. B, Clonogenetic assay of CD34+ cells plated in semisolid erythroid or monocytic medium in the absence (Untreated) or in the presence of 100 ng/ml TWEAK (TWEAK) or 5 µg/ml anti-Fn14 (ITEM-1). Data are expressed as number of burst-forming unit erythroid (BFU-E) and colony-forming unit macrophage (CFU-M) colonies per 500 plated cells. The results are the mean ± SD of three independent experiments. *, p < 0.05 vs control. C, Photomicrographs of BFU-E colonies cultivated in semisolid erythroid medium in the absence (Untreated) or in the presence of either anti-Fn14 (ITEM-1) or TWEAK (TWEAK).

 
IFN-{gamma}-mediated inhibition of erythropoiesis is mediated by TWEAK, TRAIL, and CD95L

The observation that IFN-{gamma} up-regulates TWEAK and TRAIL expression on immature erythroid cells suggests that TNF family ligands may contribute to IFN-{gamma}-induced suppression of erythropoiesis through the inhibition of proliferation and differentiation or induction of apoptosis (5, 6). To investigate whether IFN-{gamma}-mediated inhibition of erythroblast proliferation was orchestrated by multiple interactions between TNF family ligands and their cognate receptors, we treated immature erythroid cells with IFN-{gamma} in the presence of Fn14:Fc fusion protein, DR5:Fc fusion protein, and NOK-1 Ab, alone or in combination, to neutralize the effects of TWEAK, TRAIL, or CD95L, respectively. Cell growth and differentiation was then evaluated after 6 additional days of unilineage erythroid culture. The antiproliferative activity of IFN-{gamma} remained almost unaffected by the single or combined neutralization of TWEAK and TRAIL and was partially reverted by the inhibition of CD95L alone or in combination with either TWEAK or TRAIL inhibition (Fig. 6A). Simultaneous treatment with Fn14:Fc, DR5:Fc, and NOK-1 completely abrogated the growth inhibitory activity of IFN-{gamma} in erythroid cells, but not in control unilineage neutrophilic cultures (Fig. 6A). Similarly, only the combined neutralization of TWEAK, TRAIL, and CD95L was able to restore survival and differentiation inhibited by IFN-{gamma} in erythroid precursors (Fig. 6, B and C). Thus, the simultaneous involvement of TWEAK, TRAIL, and CD95L mediates the inhibitory action of IFN-{gamma} on the growth, differentiation and viability of immature erythroid precursors, defining a new role for TWEAK and TRAIL as mediators of the anti-erythropoietic effects of IFN-{gamma}.



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  		FIGURE 6. The combined inhibition of TWEAK, TRAIL, and CD95L abrogates IFN-{gamma}-mediated suppression of erythropoiesis. A, Hemopoietic progenitors at day 5 of erythroid or neutrophilic differentiation were cultivated in standard unilineage medium in the presence of 300 U/ml IFN-{gamma} with or without 5 µg/ml Fn14:Fc, DR5:Fc, or NOK-1. The percentage of protection was assessed at day 11 for erythroid cultures and at day 17 for neutrophilic cultures as described in Materials and Methods. Mouse IgG1 was used as a control for Fc and NOK-1 binding. B, Differentiation analysis of erythroblasts untreated (Control) or treated for 6 days either with 300 U/ml IFN-{gamma} (IFN-{gamma}) or with IFN-{gamma} and inhibitors of TWEAK, TRAIL, and CD95L (IFN-{gamma} + Fn14:Fc + DR5:Fc + NOK-1). Data are the mean ± SD of four independent experiments with cells from different donors. C, Percentage of apoptotic cells in erythroblast cultures untreated (Control) or treated as in A. At day 11, cells were harvested and stained with annexin V/propidium iodide as described in Materials and Methods. Data are the mean ± SD of three independent experiments performed with cells from different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Homeostasis of the hemopoietic compartment has been shown to involve not only cell proliferation and differentiation but also programmed cell death or apoptosis (38). Components of the apoptotic machinery, such as death receptors/ligands and caspases, are likely to play a key role in balancing hemopoietic cell production in vivo, as suggested by the dysregulated extramedullary expansion of myeloid progenitor cells that occurs in mice deficient either for CD95 or its ligand (39). The role of death receptors and their ligands in hemopoietic homeostasis is particularly evident during the development of erythroid cells. In vitro studies on primary human cells have shown that both CD95L and TRAIL are involved in the negative control of erythropoiesis (5, 7), suggesting that death receptor activation may act as a feedback mechanism that inhibits unnecessary erythroblast production by activating caspases in immature erythroid precursors (6). The importance of death receptor/ligand interactions in the regulation of erythropoiesis has been confirmed by elegant studies on transgenic zebrafish that express a dominant negative form of the hemopoietic death receptor ZH-DR and display severe erythrocyte accumulation (40). Likewise, a diffused erythrocytosis is found in mice embryos deficient for caspase-8 or FADD that represent the common upstream effectors of intracellular signaling pathways activated by death receptors (41, 42).

TWEAK is a member of the TNF family of cytokines involved in inflammation, angiogenesis, and cytotoxicity against tumor cells. Like other TNF family members, TWEAK seems to play different roles depending on the cellular context, ranging from apoptosis induction in transformed cells to stimulation of growth and migration in endothelial cells. Fn14 was recently identified as a fully functional TWEAK receptor (27). This small membrane protein is able to mediate a variety of intracellular signals in a cell-specific fashion including apoptosis, necrosis, proliferation, and survival (17, 22, 31, 43). Proliferative signals elicited by Fn14 are likely to be mediated by association of TNFR-associated factor proteins with the Fn14 cytoplasmic domain and subsequent stimulation of NF-{kappa}B activity (29, 30), whereas survival signals involve NF-{kappa}B-mediated up-regulation of Bcl-XL and Bcl-W expression (44). Death signals initiated by Fn14 have been thoroughly studied in tumor cell lines and have been shown to involve caspase activation as well as cathepsin B-dependent necrosis (31). However, since the Fn14 cytoplasmic region does not contain a death domain, it is not clear whether one or more accessory proteins are required to initiate TWEAK-mediated cell death. The physiological significance of TWEAK/Fn14 interactions is largely unknown. We have found that Fn14 is expressed on erythroid precursor cells, which are characterized by a high proliferative potential. This finding is consistent with previous reports on Fn14 expression in actively dividing tissues, such as the regenerating liver and injured vessel wall (27, 45), thus suggesting a general role for Fn14 in balancing cell expansion. TWEAK mRNA is expressed in erythroid precursor cells until the latest maturation stages, whereas TWEAK protein is present at low levels on the plasma membrane. It is currently unknown to which extent TWEAK contributes to the modulation of erythroid differentiation. Indeed, a potential role of TWEAK in the physiological regulation of erythropoiesis may be masked in vivo by a functional redundancy among TWEAK, CD95L, and TRAIL. However, we showed that the increase in TWEAK expression induced by IFN-{gamma} results in the generation of anti-erythropoietic signals, thus suggesting that the TWEAK/Fn14 system may effectively contribute to the erythroid suppression that occurs in pathological conditions.

IFN-{gamma} inhibits erythropoiesis in vitro and in vivo and has been implicated in the increased apoptotic response displayed by hemopoietic cells of patients with bone marrow failure syndromes (13, 16, 35, 46). In aplastic anemia, IFN-{gamma} overexpression in the marrow microenvironment has been suggested to play a role in the pathogenesis of hemopoietic suppression (16). IFN-{gamma} is also overexpressed by bone marrow mononucleated cells derived from Fanconi anemia patients (47) where it may contribute to the pathogenesis of erythroid failure. IFN-{gamma} induces CD95 expression on CD34+ hemopoietic progenitors and suppresses the growth and clonogenetic potential of long term culture-initiating cells (15). Moreover, IFN-{gamma} has been shown to up-regulate the expression of CD95 and apoptotic caspases in progenitor cells of the erythroid lineage (14, 35), suggesting that it may act by sensitizing immature erythroblasts to negative modulators of hematopoiesis. TNF family members can inhibit red cell production by inducing either growth and differentiation arrest or apoptosis (5, 6). Following treatment of erythroblast cultures with IFN-{gamma}, we have observed both induction of cell death and inhibition of proliferation and differentiation, which altogether are likely to account for IFN-{gamma}-mediated suppression of erythropoiesis. The results presented in this study show that TWEAK and TRAIL are involved in IFN-{gamma}-mediated inhibition of erythropoiesis and that only the combined neutralization of TWEAK, TRAIL, and CD95L can restore growth and differentiation of IFN-{gamma}-treated erythroid precursor cells. In line with these observations, it would be important to investigate whether TWEAK and TRAIL are overexpressed in conditions of erythroid suppression in vivo and to assess their functional involvement in bone marrow failure syndromes. The accomplishment of these studies may shed light on the mechanisms of IFN-{gamma}-mediated hemopoietic suppression and contribute to the understanding of immune-mediated disorders of red cell production.


   Acknowledgments
 
We thank P. Di Matteo for excellent technical assistance and G. Loreto for graphics. We are grateful to Drs. Hideo Yagita, Henning Walczak, and Jan Paul Medema for generously providing reagents.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Italian Association for Cancer Research, the Ministero dell’Istruzione e dell’Università, and the National Institutes of Health (Grant HL-39727 to J.A.W). Back

2 N.F. and F.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Ruggero De Maria, Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, viale Regina Elena 299, Rome, 00161, Italy. E-mail address: rdemaria{at}tin.it Back

4 Abbreviations used in this paper: Epo, erythropoietin; DR, death receptor; Fn14, fibroblast growth factor-inducible 14; CD95L, CD95 ligand. Back

Received for publication August 10, 2004. Accepted for publication May 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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