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Keywords:

  • erythropoiesis;
  • erythroid burst-forming units (BFU-E);
  • CD34+ cells;
  • FLIP;
  • inflammatory cytokines

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

Summary. In this study, quiescent bone marrow-derived CD34+ erythroid burst-forming units (BFU-E) were found to be resistant to the inhibitory effects of tumour necrosis factor (TNF)-α and -β as well as interferon (IFN)-α, -β and -γ, in contrast to those stimulated by a combination of erytrhropoietin (Epo) plus kit ligand (KL). Unexpectedly, we found that TNF-α also inhibited the apoptosis of quiescent normal human CD34+ BFU-E cells. Accordingly, TNF-α added to CD34+ cells cultured for 2 d in serum-free medium protected clonogeneic BFU-E from undergoing serum deprivation-mediated apoptosis. Furthermore, the prosurvival effect of TNF-α in quiescent CD34+ cells was consistent with its ability to induce phosphorylation of mitogen-activated protein kinase (MAPK) p42/44. However, when added to CD34+ cells that were stimulated by Epo + KL, TNF-α induced apoptosis and inhibited proliferation of BFU-E. To explain this intriguing differential sensitivity between unstimulated CD34+ cells versus those stimulated by Epo + KL, we examined the expression of apoptosis-regulating genes (FLIP, BCL-2, BCL-XL, BAD and BAX) in these cells. Of all the genes tested, FLIP became rapidly downregulated in CD34+ cells 24 h after stimulation with Epo + KL, suggesting that it may protect quiescent CD34+ BFU-E progenitors residing in the bone marrow from the inhibitory effects of inflammatory cytokines. Thus, we hypothesize that cycling cells may become more sensitive to proapoptotic stimuli (e.g. chemotherapy, inhibitory cytokines) than quiescent ones because of the downregulation of protective FLIP.

Growth factors and cytokines influence the biology of target cells by activating appropriate signalling pathways. Studies performed on immortalized human cell lines suggest that cell proliferation is regulated mainly by activation of the mitogen-activated protein kinase (MAPK) p42/44 (Sui et al, 1998) or JAK-STAT (Janus kinase signal transducers and activators of transcription; Rane & Reddy, 2002) and inhibition of apoptosis by activation of phosphatidylinositol-3 kinase (PI-3K)–serine-threonine kinase (AKT) axis (Khwaja, 1999; Kashii et al, 2000; Zhou et al, 2000) or JAK-STAT protein pathways (Teglund et al, 1998). We have reported previously that erythropoietin (Epo) in combination with kit ligand (KL) [each of which stimulates the formation of large erythroid burst-forming unit (BFU-E)-derived colonies in vitro] (Ratajczak et al, 1992, 1994, 1997, 1998a, b) activates the MAPK p42/44 and PI-3K–AKT axes and JAK-STAT proteins in normal human erythroblasts (Ratajczak et al, 2001).

The growth of erythroid colonies may be negatively affected by several ‘inhibitory’ cytokines such as tumour necrosis factor-α (TNF-α), tumour necrosis factor-β (TNF-β), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ) and transforming growth factor-β1 (TGF-β1) (Majka et al, 2000a; Golstein & Wyllie, 2001; Giron-Michel et al, 2002; Verma et al, 2002; Chung et al, 2003). Serum levels of these cytokines are elevated in patients suffering from chronic infections (Means & Krantz, 1992; Weiss, 2002), and thus all these cytokines have been implicated in the pathogenesis of anaemia in chronic disorders (ACD). The presence of cell surface receptors for TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ and TGF-β1 on CD34+ and differentiating erythroblasts has been demonstrated by several investigators (Majka et al, 2000a; Golstein & Wyllie, 2001; Janowska-Wieczorek et al, 2001; Giron-Michel et al, 2002; Verma et al, 2002; Chung et al, 2003). We also demonstrated that several inhibitory cytokines are expressed at the mRNA level in early erythroid precursors (Janowska-Wieczorek et al, 2001; Majka et al, 2001; Ratajczak et al, 2003). Some of them, such as TGF-β for example, are also detectable at the protein level in conditioned media harvested from these cells (Janowska-Wieczorek et al, 2001; Majka et al, 2001; Ratajczak et al, 2003). This suggests the potential existence of negative autocrine/paracrine regulatory loops that regulate the development of erythroid cells in bone marrow (Janowska-Wieczorek et al, 2001; Majka et al, 2001; Ratajczak et al, 2003). and also implies that some protective mechanisms operating at the level of early erythroid progenitors have to exist in order to counterbalance the potential inhibitory/proapoptotic signals triggered by these negative regulatory loops.

It has been demonstrated recently that human CD34+ haematopoietic stem/progenitor cells express high levels of FLICE [FADD (FAS-associated death domain protein)-like interleukin-1 beta-converting enzyme] inhibitory protein (FLIP), which is the inactive homologue of caspase-8 (Kim et al, 2002). High expression levels of FLIP probably provide one level of protection for CD34+ cells from death receptor-mediated apoptosis (TNFR1 p55 and CD95/APO-1) (Kim et al, 2002). Moreover, it has been shown that signalling from TNFR1 p55 and CD95/APO-1 may lead in some cell types to a paradoxical stimulation of proliferation and inhibition of apoptosis if FLIP becomes activated in these cells instead of caspase-8 (Budd, 2002).

In this study, we focused on the influence of erythroid ‘inflammatory’ cytokines on the phosphorylation of major proliferative and prosurvival signalling pathways in normal human CD34+ cells and erythroblasts (MAPK p42/44, AKT and STAT proteins). We hypothesize that identification of these pathways could have important implications for the development of new therapeutic strategies for ACD patients that are based on modulating signalling in early erythroid progenitor/precursor cells.

In our studies, performed on purified normal human CD34+ cells, we found that several inflammatory cytokines did not affect the survival of quiescent CD34+ erythroid progenitors, but this changed when these cells were stimulated to proliferate/differentiate along the erythroid lineage with Epo + KL. We demonstrated that this differential sensitivity of BFU-E progenitors was related to the higher expression of protective FLIP in unstimulated early erythroid progenitors which, however, were rapidly downregulated in cells stimulated with Epo + KL.

Human CD34+ cells.  Light-density bone marrow mononuclear cells (BM MNC) were obtained from consenting healthy donors, depleted of adherent cells and T lymphocytes (AT MNC) and enriched for CD34+ cells by immunoaffinity selection using MiniMACS paramagnetic beads (Miltenyi Biotec, Auburn, CA, USA) as described previously (Majka et al, 2000b, 2002). The purity of isolated BM CD34+ cells was > 95% as determined by fluorescence-activated cell sorter (FACS) analysis.

Ex vivo expansion of human erythroid cells.  CD34+ cells were expanded in a serum-free liquid system as described previously (Ratajczak et al, 1998a,b; Majka et al, 2001). Briefly, CD34+ AT MNC were resuspended in Iscove's Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY, USA) 104/ml supplemented with 25% artificial serum containing 1% delipidated, deionized and charcoal-treated bovine serum albumin (BSA), 270 µg/ml iron-saturated transferrin, insulin (20 µg/ml) and 2 mmol/l l-glutamine (all from Sigma, St Louis, MO, USA). BFU-E growth was stimulated with recombinant human (rh) Epo (2 U/ml) and rh KL (10 ng/ml). Cytokines were obtained from R & D Systems, Minneapolis, MN, USA. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. After 14 d under these conditions, approximately 100% of the expanded cells were glycophorin A (GPA-A) positive and CD33 and glycoprotein (gp)IIa/IIIb negative. In our experiments, we used erythroid cells expanded for 8–10 d as described above.

Exposure to proinflammatory cytokines.  Freshly isolated BM MNC CD34+ cells or erythroblasts that had been expanded for 11 d were exposed for 24–48 h in serum-free medium to the following proinflammatory cytokines (all from R & D Systems): TNF-α and TNF-β at a dose of 5–50 ng/ml, IFN-α, IFN-β and IFN-γ at a dose of 10–500 U/ml and TGF-β1 AT 1–5 ng/ml.

Detection of apoptosis.  Apoptosis was assessed by staining cells with fluorescein isothiocyanate (FITC)–Annexin V followed by flow cytometric analysis (FACScan; Becton Dickinson, Mountain View, CA, USA) and using the apoptosis detection kit (R & D Systems) according to the manufacturer's protocol. Activation of caspase-3 was determined by FACS, according to the manufacturers' protocols (BD Pharmingen, San Diego, CA, USA) (Ratajczak et al, 2001; Majka et al, 2002).

Real-time reverse transcription polymerase chain reaction (RT-PCR).  For analysis of FLIP mRNA levels, total mRNA was isolated from cells with the RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse-transcribed with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA). Detection of FLIP and β-actin mRNA levels was performed by real-time RT-PCR assay using an ABI Prism® 7000 sequence detection system (ABI, Foster City, CA, USA). A 25-µl reaction mixture contained 12·5 µl of SYBR Green PCR master mix, 10 ng of cDNA template, 5′-CATGGGAGATTCATGCCCTTA-3′ forward and 5′-TGGCCCTCTGACACCACATA-3′ reverse primers for FLIP and 5′-GGATGCAGAAGGAGATCACTG-3′ forward and 5′-CGATCCACACGGAGTACTTG-3′ reverse primers for β-actin. Primers were designed with primer express software. The threshold cycle (Ct), i.e. the cycle number at which the amount of amplified gene of interest reaches a fixed threshold, was subsequently determined. Relative quantification of c-MET mRNA expression was calculated by the comparative Ct method. The relative quantification value of target, normalized to an endogenous control β-actin gene and relative to a calibrator, was expressed as 2–ΔΔCt (fold), where ΔCt = Ct of the target gene (FLIP)–Ct of endogenous control gene (β-actin), and ΔΔCt = ΔCt of samples for the target gene–ΔCt of the calibrator for the target gene.

Phosphorylation of intracellular pathway proteins.  Western blots were performed using extracts prepared from haematopoietic cells (1 × 106) that were kept in Roswell Park Memorial Institute (RPMI) medium containing low levels of BSA (0·5%) to render the cells quiescent. The cells were then divided and stimulated with optimal doses of Epo, KL or inflammatory cytokines for 5 min or 10 min at 37°C. The cells were then lysed for 10 min on ice in M-Per lysing buffer (Pierce, Rockford, IL, USA) containing protease and phosphatase inhibitors (Sigma). Subsequently, the extracted proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and the fractionated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) as described previously (Majka et al, 2001, 2002; Ratajczak et al, 2001). Phosphorylation of each of the intracellular kinases, 44/42 MAPK and AKT, and STAT-1–6 proteins was detected using commercial mouse phosphospecific monoclonal antibody (mAb) (p44/42) or rabbit phosphospecific polyclonal antibodies for each of the remainder (all from New England Biolabs, Beverly, MA, USA) with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (IgG) or goat anti-rabbit IgG as a secondary antibody (Santa Cruz Biotech, Santa Cruz, CA, USA), as described. Equal loading in the lanes was evaluated by stripping the blots and reprobing with appropriate mAbs: p42/44 anti-MAPK antibody clone #9102, anti-AKT antibody clone #9272 and anti-STAT 3 #9132 (New England Biolabs), anti-STAT 1 #sc-464, anti-STAT 6 #sc-1689 (Santa Cruz Biotech) and anti-STAT 5 #89 (Transduction Laboratories, Lexington, KY, USA). The membranes were developed with an enhanced chemiluminescence reagent (Amersham Life Sciences, Little Chalfont, UK) and subsequently dried and exposed to film (HyperFilm, Amersham Life Sciences). Densitometric analysis was performed using exposures that were within the linear range of the densitometer (Personal Densitometer SI; Molecular Dynamics, Sunnyvale, CA, USA) and imagequant software (Molecular Dynamics).

Detection of FLIP by Western blot.  Cell lysates from CD34+ cells that had been stimulated or not for 24 h with Epo + KL were prepared as described above. FLIP protein was detected using anti-FLIP polyclonal rabbit IgG (Upstate, Charlottesville, VA, USA) detected with goat anti-rabbit IgG as a secondary antibody (Santa Cruz Biotech) and anti-rabbit HRP-conjugated mAb according to the manufacturer's protocol. The FLIP antibody used in this study detects both long and short forms of FLIP.

Statistical analysis.  Arithmetic means and standard deviations were calculated on a MacIntosh computer using instat 1.14 (GraphPad, San Diego, CA, USA) software. Data were analysed using the Student t-test for unpaired samples. Statistical significance was defined as P < 0·05.

Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

Several erythropoietic inflammatory cytokines such as TNF-α, TNF-β, IFN-β and IFN-γ, but not IFN-α or TGF-β1, increased the survival of unstimulated BM-derived CD34+ cells. Accordingly, if these cells were plated in a serum-free medium without growth factors, a decrease in activation of caspase-3 was observed using intracellular staining (Fig 1A) and the Annexin-V binding assay (Fig 1B) of purified CD34+ cells. As expected, this prosurvival effect was also observed in the presence of KL (positive control). However, Epo alone did not affect the survival of CD34+ cells (Fig 1).

image

Figure 1. Normal human CD34+ cells were plated in serum-free medium in the absence or presence of various cytokines. After 48 h, cells were evaluated by FACS for the presence of activated intracellular caspase-3 (A) and for their ability to bind Annexin V (B). We observed that apoptosis of CD34+ cells in addition to KL was also slightly inhibited by TNF-α, TNF-β, IFN-β and IFN-γ. The experiment was repeated four times with similar results. A representative result is shown.

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TNF-α increases the survival of human quiescent CD34+ BFU-E

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

As clonogenic BFU-E comprise only about 1–2% of this population of CD34+ cells (Krause et al, 1996), to learn more about the effects of inflammatory cytokines on the survival of quiescent BFU-E, we used a different type of assay. We cultured normal purified bone marrow-derived CD34+ cells under liquid serum-free conditions for 48 h in the absence or presence of known erythropoietic inhibitory factors such as TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ and TGF-β1. Subsequently, cells were washed out from those cytokines and replated into secondary semi-solid cultures before stimulation with Epo + KL to grow erythroid colonies (Fig 2).

image

Figure 2. Normal quiescent CD34+ cells were cultured for 48 h in the absence (–) or presence of TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ or TGF-β1. Subsequently, cells were washed out from these inhibitory cytokines and plated in methylcellulose cultures to grow erythroid colonies. Erythroid colonies were counted 14 d later under an inverted microscope. The experiment was repeated three times with similar results. The number of colonies grown from cells incubated with no cytokines (–) is shown as 100%. The number of BFU-E colonies formed by freshly isolated cells was 557 ± 68% compared with cells cultured for 48 h in serum-free medium (–). *P < 0·000001 and **P < 0·01 compared with cells cultured in medium alone.

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To our surprise, every inflammatory cytokine evaluated in this study, except for TGF-β1, failed to decrease survival of quiescent clonogenic BFU-E progenitors, as shown by their ability to grow erythroid colonies after replating to methylcellulose cultures. Moreover, TNF-α strongly enhanced the survival of clonogenic CD34+ BFU-E (P < 0·000001). Accordingly, the number of colonies formed by CD34+ cells cultured for 48 h in the presence of TNF-α was similar to that grown by freshly isolated CD34+ cells (Fig 2). A less pronounced effect was observed in the presence of TNF-β (P < 0·01).

Of note, similar results were obtained for clonogenic granulocyte–macrophage colony-forming units (CFU-GM and megakaryocyte CFU (CFU-Meg) progenitors (not shown) – unstimulated or stimulated with granulocyte–macrophage colony-stimulating factor (GM-CSF) + KL and thrombopoietin + KL, respectively, suggesting that this mechanism operates at the level of other clonogenic progenitors.

Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

To explain our in vitro data, we analysed the activation of signalling pathways that are potentially related to survival/proliferation in normal CD34+ cells after exposure to erythropoietic inhibitory cytokines. To our surprise, we found that TNF-α and TNF-β, like KL, stimulated phosphorylation of MAPK p42/44 (Fig 3A). At the same time, however, serine-threonine kinase (AKT), which is crucial for cell survival, was phosphorylated in CD34+ cells by KL only (Fig 3A). Of note, none of the inflammatory cytokines evaluated in this study negatively affected KL-mediated phosphorylation of AKT in normal human CD34+ BM MNC if added 15 min before or at the time of KL stimulation (not shown).

image

Figure 3. Normal human CD34+ cells were made quiescent (24-h culture in serum-free medium supplemented with 0·5% BSA) and then stimulated with Epo, KL, IFN-α, IFN-β, IFN-γ, TNF-α and TNF-β. (A) Phosphorylation of MAPK p42/44 and AKT; (B) phosphorylation of STAT-5; and (C) phosphorylation of STAT-3 proteins. We found that TNF-α and TNF-β stimulated phosphorylation of MAPK p42/44 and IFN-α, -β and -γ strongly stimulated the phosphorylation of STAT-5 and STAT-3 proteins. The experiment was repeated three times with similar results.

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Again unexpectedly, we found that IFN-α, IFN-β and IFN-γ strongly stimulated phosphorylation of STAT-5 and STAT-3 proteins in CD34+ cells (Fig. 3B and C). However, phosphorylation of STAT-5 and STAT-3 proteins was not affected after stimulation by TNF-α and TNF-β. TGF-β1 did not influence the phosphorylation of MAPK p42/44, AKT or STAT proteins in CD34+ cells (not shown).

Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

The experiment was repeated in conditions in which CD34+ cells were cultured for 48 h in serum-free medium supplemented with the optimal combination of erythropoietic stimulators (Epo + KL) in the presence or absence of erythropoietic inhibitory cytokines (Fig 4). We found that all these factors, when added to the suspension of CD34+ cells stimulated by Epo + KL, inhibited their ability to grow BFU-E after these cells were replated onto secondary methylcellulose-supplemented cultures (Fig 4).

image

Figure 4. Normal CD34+ cells were cultured for 48 h in serum-free medium supplemented with Epo + KL in the absence (–) or presence of TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ or TGF-β1. Subsequently, cells were washed out from these inhibitory cytokines and plated in methylcellulose cultures to grow erythroid colonies. Erythroid colonies were counted 14 d later under an inverted microscope. The experiment was repeated three times with similar results. The number of colonies grown in cells incubated with no inflammatory cytokines (–) is shown as 100%. *P < 0·00001 compared with cells preincubated in medium supplemented with Epo + KL alone.

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To explain this intriguing differential sensitivity between quiescent CD34+ cells (Fig 2) versus CD34+ cells stimulated by Epo + KL (Fig 4), we examined the expression of apoptosis-regulating genes (FLIP, BCL-2, BCL-XL, BAD and BAX) in quiescent versus Epo + KL-stimulated cells. We found, using real-time RT-PCR, that of all the genes tested, only FLIP became rapidly downregulated in CD34+ cells 24 h after stimulation with Epo + KL (Fig 5A). This decrease in FLIP expression was subsequently confirmed at the protein level using Western blotting (Fig 5B).

image

Figure 5. (A) FLIP mRNA is downregulated (real-time RT-PCR) in normal human CD34+ cells stimulated by Epo + KL. The experiment was repeated three times, and the data are pooled together. *P < 0·0001 compared with quiescent non-stimulated CD34+ cells. (B) Western blot analysis of FLIP expression in lysates from normal and Epo + KL-stimulated CD34+ cells. The experiment was repeated three times with similar results. A representative result is shown.

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Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

Finally, we repeated our experiments on normal human erythroblasts that had been expanded in serum-free cultures from a population of CD34+ cells. As predicted, none of these inflammatory cytokines protected ex vivo-expanded d-9 erythroblasts from undergoing apoptosis as demonstrated by Annexin V binding assay (Fig 6).

image

Figure 6. Normal human erythroblasts were plated in serum-free medium in the absence or presence of various cytokines. After 48 h, cells were evaluated by FACS for their ability to bind Annexin V. The apoptosis of erythroblasts was inhibited by KL and Epo and not affected by TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ and TGF-β1. The first graph (–) demonstrates cells cultured without any growth factors or cytokines. The experiment was repeated four times with similar results. A representative study is shown.

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However, THE signal transduction studies performed on these cells (Fig 7) revealed that several signalling pathways that are considered to play an important role in stimulating cell proliferation or increasing cell survival became unexpectedly activated in normal human erythroid cells after stimulation by inflammatory cytokines. Accordingly, although TNF-α again slightly stimulated phosphorylation of MAPK p42/44, IFN-α, IFN-β and IFN-γ strongly stimulated phosphorylation of STAT-1, -3, -5 and -6 proteins (Fig 7). Moreover, as reported in our previous study (Ratajczak et al, 2001), KL strongly phosphorylated AKT, MAPK p42/44 and STAT proteins in human erythroblasts (Fig 7).

image

Figure 7. Western blot data of normal human erythroblasts stimulated by various cytokines. (A) Phosphorylation of AKT (top) and MAPK p42/44 (bottom). (B) Phosphorylation of STAT 1–6 proteins. IFN-α, -β and -γ strongly phosphorylated STAT-1, -3, -5 and -6 proteins in these cells. The experiment was repeated three times with similar results. A representative study is shown.

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Finally, none of the inflammatory cytokines evaluated in this study negatively affected KL-mediated phosphorylation of AKT, MAPK p42/44 and STAT proteins in normal human erythroblasts if added 15 min before or at the time of KL stimulation (not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References

The molecular mechanisms that regulate the development of erythroid cells are still not fully understood. In vitro data in serum-free conditions have shown that the combination of Epo and KL is the most potent developmental stimulator of haemoglobinized large erythroid colonies (Ratajczak et al, 1992, 1994, 1997, 1998a,b, 2001). In our previous study, we found that, in human CD34+ cells and erythroblasts, erythropoietic factors may activate both similar and different signalling pathways (Ratajczak et al, 2001). Activation of the JAK-STAT, MAPK p42/44 or PI-3K-AKT axes alone is not sufficient either to stimulate cell proliferation or to inhibit apoptosis, suggesting that these processes are regulated by the orchestrated activation of multiple signalling cascades (Ratajczak et al, 2001). Accordingly, we found that, although cell proliferation was more related to simultaneous activation of JAK-STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI-3K-AKT, MAPK p42/44 and JAK-STAT proteins. Involvement of these pathways in erythropoiesis was confirmed by blocking the PI-3K–AKT axis by the Ly290042 inhibitor (Haseyama et al, 1999; Myklebust et al, 2002) or by perturbing the JAK-STAT pathway by gene knock-outs (Oda et al, 1998; Socolovsky et al, 2001). Accordingly, although inhibition of the PI-3K axis leads to a decrease in survival of early erythroid cells (Haseyama et al, 1999; Myklebust et al, 2002; Bouscary et al, 2003), mice with STAT-5 knock-out display severe defects in the survival of erythroid progenitors (Socolovsky et al, 2001).

Erythropoiesis is inhibited in vitro by several inhibitory cytokines, the levels of which are elevated in the sera of patients suffering from chronic infections and may thus play an important role in vivo in the pathogenesis of anaemia from chronic disorders (Means & Krantz, 1992; Weiss, 2002). The most important erythropoietic inhibitors are TNF-α, TNF-β, IFN-α, IFN-β, IFN-γ and TGF-β1. The suppressive effect of TGF-β and IFN-α and -β on erythropoiesis has been attributed to activation of MAPK p38 and suppressors of cytokine signalling (SOCS) proteins (Giron-Michel et al, 2002; Verma et al, 2002). As signalling pathways activated in normal erythroid progenitor and precursor cells have not been investigated in detail, this prompted us to perform such an analysis.

We report some unexpected results. First, we observed that quiescent CD34+ erythroid BFU-E progenitors were resistant to inhibition by TNFs and IFNs and, to our surprise, TNF-α strongly protected early quiescent CD34+ erythroid BFU-E progenitor cells from undergoing apoptosis. At the same time, however, all the inflammatory cytokines evaluated in our study, including TNF-α, inhibited BFU-E growth when added to CD34+ cells that had been stimulated by Epo + KL. As quiescent CD34+ cells express a high level of FLIP (Kim et al, 2002), which, as shown in this study, was rapidly downregulated in CD34+ cells stimulated by Epo + KL, we postulate that FLIP may protect quiescent CD34+ BFU-E progenitors residing in the bone marrow from the potentially inhibitory effects of autocrine/paracrine-secreted inflammatory cytokines. We reported that several of these factors are secreted in the bone marrow microenvironment by early erythroid cells (Janowska-Wieczorek et al, 2001; Majka et al, 2001; Ratajczak et al, 2003). In addition, it has also been reported that caspase-3 is transiently activated during the early stages of erythropoiesis in order to ensure proper transition from the erythroblast to the reticulocyte stage (Zermanti et al, 2001). A transient ‘controlled’ activation of caspase-3 in maturing erythroid cells is probably necessary for the cleavage of proteins involved in nucleus integrity (laminin B) and chromatin condensation (acinus) without inducing cell death and cleavage of GATA-1 (Zermanti et al, 2001).

FLIP is homologous to caspase-8 in containing two death domains that can interact with the corresponding domain of FADD, but its caspase domain is non-functional (Majka et al, 2000b; Budd, 2002; Kim et al, 2002; Chung et al, 2003). FLIP was recently implicated to play an important role in mediating unexpected antiapoptotic signals from activated FAS (CD95/APO-1) and TNF-receptor 1 (p55 TNFR1). Thus, FLIP is probably a switch molecule that diverts death receptor signals from cell death to proliferation (Budd, 2002). In support of this hypothesis, FLIP has recently been reported to play an important role in the resistance of CD34+ cells to Fas-mediated apoptosis (Kim et al, 2002) as well as in KL-mediated resistance of erythroid cells to IFN-γ (Budd, 2002). Furthermore, the erythroid differentiation sensitized K-562 human erythroleukaemia cells to TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis by downregulating cellular (c)-FLIP (Hietakangas et al, 2003).

As FLIP-mediated signalling from TNF-R1 involves activation of MAPK p42/44 (Budd, 2002), this may explain why TNF-α has been shown to stimulate, for example, the growth of T and B lymphocytes, vascular smooth cells and dendritic cells (Jelinek & Lipsky, 1987; Yokota et al, 1988; Larsen et al, 1990). Based on this, we propose that a similar antiapoptotic mechanism to TNF-α also operates at the level of quiescent CD34+ BFU-E and is related to MAPK p42/44 activation by FLIP. Further studies, however, are required to confirm a protective role of FLIP in which intracellular expression of FLIP would be directly downregulated in normal erythroid cells using (1) antisense oligodeoxynucleotides against FLIP mRNA; or (2) a double-stranded RNA-mediated interference (RNAi) strategy (Gewirtz et al, 1998; Martinez et al, 2003).

Interestingly, we noticed that the IFNs, which strongly induced apoptosis in proliferating early erythroid cells, strongly activated the phosphorylation of STAT proteins at the same time. In accordance with this is the fact that activation of Jak1-STAT1 proteins has recently been demonstrated in normal erythroid cells stimulated by IFN-α and IFN-β (Giron-Michel et al, 2002). Our data show that not only IFN-α and IFN-β but also IFN-γ are strong inducers of STAT protein phosphorylation in normal human erythroid cells.

It has been postulated that STAT-5 plays an important prosurvival role in normal early erythroid cells (Oda et al, 1998; Teglund et al, 1998; Ward et al, 1999; Socolovsky et al, 2001). However, despite the fact that IFNs efficiently activated the STAT-5 protein in erythroid cells, they induced apoptosis, which indicates that activation of STAT proteins alone is probably not sufficient to protect erythroid cells from undergoing apoptosis. Similarly, in our previous study, we demonstrated that the PI-3K–AKT axis, which plays an important antiapoptotic role in early erythroid cells (Haseyama et al, 1999; Myklebust et al, 2002; Bouscary et al, 2003), was activated in both CD34+ cells and early erythroblasts by KL. Furthermore, we noticed that this potential prosurvival signalling by KL was not negatively affected at the time of KL stimulation by any of the inflammatory cytokines used in our studies. The fact that TNF-α did not affect phosphorylation of AKT, yet still had a strong antiapoptotic effect on quiescent CD34+ cells, suggests that, in addition to the PI-3K–AKT pathway, other pathways such as MAPK p42/44 may also play an important role in increasing the survival of quiescent CD34+ cells. This further supports the notion that, in contrast to established haematopoietic cell lines, proliferation and survival of primary human haematopoietic cells is regulated by several complementary pathways (Ratajczak et al, 2001).

In conclusion, our data suggest that the downregulation of FLIP may cause cycling BFU-E cells to become more sensitive to proapoptotic stimuli (e.g. chemotherapy, inhibitory cytokines) than quiescent cells. We also postulate that quiescent CD34+ erythroid progenitor cells are somehow autoprotected by FLIP from the potential inhibitory influence of certain autocrine/paracrine loops involving inhibitory cytokines. Finally, we hypothesized that the molecular strategies aimed at increasing the level of FLIP in erythroid progenitors could potentially increase their resistance to those inflammatory cytokines that play a crucial role in the pathogenesis of anaemia in chronic disorders.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Several erythropoietic inhibitory cytokines inhibit apoptosis of quiescent CD34+ cells
  6. TNF-α increases the survival of human quiescent CD34+ BFU-E
  7. Effects of erythropoietic inflammatory cytokines on the activation of signalling pathways that are related to cell survival in quiescent CD34+ cells
  8. Effect of erythropoietic inhibitory cytokines on CD34+ cells stimulated by Epo + KL
  9. Erythropoietic inhibitory cytokines affect survival of normal human erythroblasts
  10. Discussion
  11. Acknowledgments
  12. References
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