Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by Civin, C. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by Civin, C. I.
Stem Cells 2002;20:174-182 www.StemCells.com
© 2002 AlphaMed Press

Human CD34+ Hematopoietic Stem/Progenitor Cells Express High Levels of FLIP and Are Resistant to Fas-Mediated Apoptosis

Heeje Kima, Katharine A. Whartenbya, Robert W. Georgantas, IIIa, John Wingardb, Curt I. Civina

a Department of Oncology and Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;
b Division of Hematology/Oncology, University of Florida, Gainesville, Florida, USA

Key Words. Fas • FLIP • Caspase • Stem cells • CD34 • Progenitor cells • Apoptosis

Curt I. Civin, M.D., Johns Hopkins Oncology Center, Bunting-Blaustein Cancer Research Building, Room 2M44, 1650 Orleans Street, Baltimore, Maryland 21231, USA. Telephone: 410-955-8816; Fax: 410-955-8897; e-mail: civincu{at}jhmi.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We sought to determine whether lympho-hematopoietic stem-progenitor cells (HSC) from human placental/umbilical cord blood (CB) or adult mobilized blood (PBSC) are sensitive to Fas-induced apoptosis. Human CD34+ cells from CB or PBSC were cultured in serum-free medium, with or without hematopoietic growth factors (FKT: FLT-3 ligand [FL], KIT ligand [KL], and thrombopoietin [TPO]), and with or without soluble Fas ligand (sFasL) or agonistic anti-Fas antibody. After 5-48 hours of culture, cells were assessed for viability and stained with Annexin V and 7-Aminoactinomycin D for apoptosis analysis by fluorescence-activated cell sorting. Cultured cells were also assessed by in vitro hematopoietic colony-forming cell (CFC) and in vivo nonobese diabetic/severe combined immunodeficient mouse engraftment potential (SEP) assays. Levels of Fas, FLICE inhibitory protein (FLIP), and Caspase 8 mRNA in CD34+ cells were determined by real-time quantitative polymerase chain reaction. Expression of FLIP was confirmed by Western blotting. No decrease in viability, CFC, or SEP was observed in CB or PBSC CD34+ cells cultured in the presence of sFasL or agonistic anti-Fas antibody. Human CB and mobilized PBSC CD34+ cells expressed high levels of FLIP, low ratios of Caspase 8:FLIP, and low levels of Fas. Thus, human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Activation of the Fas pathway produces apoptosis in sensitive cells, thus regulating the homeostasis of many cell types. Binding of Fas ligand (FasL; CD178) to Fas (receptor; CD95) initiates a cascade of events, ultimately activating effector caspases and leading to cellular apoptosis [1–12]. FasL, a transmembrane protein with restricted cellular expression, can be cleaved from cells by a ubiquitous metalloprotease(s), generating an agonistically effective soluble fragment (sFasL) [12–17]. Either membrane FasL on an attacking cell or the (less potent) sFasL can bind to the cognate receptor, Fas, on a target cell. Fas exists on the cell membrane as a preformed transmembrane homotrimer, and one molecule of FasL binds to each subunit of the Fas trimer. The Fas trimer can be part of a nonameric death-inducing signaling complex (DISC) consisting of three monomers each of Fas, Fas-associated death domain, and pro-caspase 8. Formation of a 12-meric DISC (including FasL) can trigger caspase-mediated apoptosis [2,18–21]. Physiologic cellular expression of the Caspase 8 inhibitor, FLICE inhibitory protein (FLIP), can block Fas-mediated apoptosis [20,22–24]. In addition, the activation of caspases is counteracted by pro-survival members of the Bcl-2 family and members of the inhibitors of apoptosis (IAP) family [25,26].

The role of the Fas pathway in early hematopoiesis is not well understood. It has been reported that some lympho-hematopoietic stem-progenitor cells (HSC) express membrane-bound FasL (mFasL) as well as Fas (receptor), which suggests autocrine and/or paracrine regulation of the Fas/FasL system in hematopoiesis [7,9,27–35]. Fas expression can be induced in bone marrow CD34+ cells from healthy individuals or acquired aplastic anemia and myelodysplastic syndrome patients by the addition of tumor necrosis factor-{alpha} or interferon-{gamma}. This suggests that the hematopoietic microenvironment may influence the susceptibility of HSC to Fas-mediated killing [7,29–35]. A number of different primary regulatory signals modulate killing of cells via the Fas pathway. For example, inhibition of HLA-DR signaling by a monoclonal antibody decreased the numbers of hematopoietic colonies generated by human CD34+ marrow cells. This effect was reduced by the addition of Fas-Ig to the medium, suggesting that the Fas pathway is involved in hematopoietic regulation [36]. Another study showed that sFasL reduced spontaneous apoptosis of ex vivo cultured fetal liver CD34+CD38- cells [27]. Growth factor deprivation induces apoptosis of HSC, and radiation-induced apoptosis was prevented by the addition of the combination of KIT ligand (KL), FLT-3 ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3). This process was shown to be mediated through the Fas pathway [25,36]. Thus, at different stages of development, or depending on milieu, HSC may exhibit differential sensitivities to Fas-mediated apoptosis, and the Fas pathway can be modulated by multiple primary regulatory signals. In addition, Fas expression has been found to be low in CD34+ human leukemia cells, which may be a mechanism of tumor cell survival [37].

The goal of the present study was to determine whether normal human CD34+ cells are susceptible to Fas-mediated apoptosis, and to investigate the basis for the susceptibility or resistance of these cells. We found that Fas pathway agonists did not induce apoptosis of CD34+ cells or reduce their in vitro hematopoietic colony-forming cells (CFC) or in vivo nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse engraftment potential (SEP). Further, we showed that human CD34+ cells expressed high mRNA and protein levels of the Fas pathway inhibitory protein, FLIP. The ratio of Caspase 8:FLIP mRNA expression in CD34+ cells was very low, similar to that in the apoptosis-resistant U266 cell line and much lower than in the apoptosis-sensitive Jurkat cell line.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Cell Cultures
Human placental/umbilical cord blood (CB) CD34+ cell preparations, purified by immunomagnetic selection following manufacturer's instructions (Miltenyi Biotechnologies; Auburn, CA; http://www.miltenyibiotec.com) and then cryopreserved, were purchased from AllCells, LLC (San Mateo, CA; http://www.allcells.com). Aliquots of cryopreserved CB CD34+ cells (approximately 106 cells) were thawed by drop-wise addition of 50 ml QBSF-60 serum-free medium (generously donated by Quality Biologicals; Gaithersburg, MD; http://www.qualitybiological.com) over 10 minutes [38,39]. After two centrifugal washes, the cell preparations were 88 ± 6% viable (by Trypan blue dye exclusion) and 95 ± 2% CD34+ (by fluorescence-activated cell sorting [FACS] analysis of immunostained cells). Fresh samples of CB were obtained from The Shands Hospital Cord Blood Bank (Gainesville, FL; http://www.shands.org) within 48 hours of collection, and purified at Johns Hopkins, as above under Institutional Review Board (IRB)-approved protocols. Mobilized blood (PBSC) CD34+ samples were obtained after cyclophosphamide and G-CSF administration from the Johns Hopkins Graft Engineering Laboratory under an IRB-approved protocol [40].

CD34+ cells were centrifuged on Ficoll-paque® (density 1.077; Amersham Pharmacia Biotech; Uppsala, Sweden; http://www.apbiotech.com) to remove dead cells, washed, then plated at 1.5 x 105 – 3 x 105 cells/ml in 24-well tissue culture plates (Becton Dickinson; San Jose, CA; http://www.bd.com) in QBSF-60 medium and incubated in a 5% CO2-humidified atmosphere at 37°C for 5 or 48 hours. Six different culture conditions were tested: A) the FKT combination of three recombinant human growth factors alone: FL (100 ng/ml), KL (100 ng/ml), and TPO (20 ng/ml) (all from PeproTech; Rocky Hill, NJ; http://www.peprotech.com) [41]; B) FKT plus 1 ng/ml recombinant human sFasL ("SUPER-FasL"; Alexis Biochemicals; San Diego, CA; http://www.alexis-corp.com); C) FKT plus 10 ng/ml sFasL; D) no FKT, no sFasL; E) no FKT, 1 ng/ml sFasL, or F) no FKT, 10 ng/ml sFasL. For the 48-hour cultures, the same quantities of FKT and sFasL were added again after the initial 24 hours of culture. Five- or 48-hour-cultured cells were assayed for viability/apoptosis, in vitro CFC, and in SEP.

Monoclonal Antibodies (mAb)
Phycoerythrin (PE)-conjugated purified mouse anti-human CD34, CD41a, CD16, and CD56, and fluorescein isothiocyanate (FITC)-conjugated purified mouse anti-human CD38, CD33, and CD71 were purchased from Becton Dickinson. FITC-conjugated purified mouse anti-human CD13 was purchased from DAKO (Carpinteria, CA; http://www.dako.dk). PE-conjugated purified mouse anti-human CD19 was purchased from Sigma (St. Louis, MO; http://www.sigmaaldrich.com). Cychrome 5 (Cy 5)-conjugated mouse anti-human CD45, and Cy 5-conjugated isotype control were purchased from Pharmingen (San Diego, CA; http://www.pharmingen.com). Annexin V-FITC was purchased from Clontech (Palo Alto, CA; http://www.clontech.com) and 7-Aminoactinomycin D (7-AAD) was purchased from Pharmingen. Anti-human Fas mAb (clone CH11) was purchased from Upstate Biotechnology (Lake Placid, NY; http://www.biosignals.com). Rat anti-FLIP mAb (clone Dave-2) was purchased from Alexis Biochemicals. Goat anti-rat immunoglobulin (Ig) G-horseradish peroxidase (HRP) secondary antibody for Western blotting was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA; http://www.jacksonimmuno.com).

Viability and Apoptosis Assays
Viability was assessed by trypan blue dye exclusion and apoptosis by Annexin V/7-AAD binding. Annexin V binds to cells at early phases of apoptosis, while 7-AAD is a DNA intercalating agent that stains only cells in the late phases of cell death when the integrity of both the cell and nuclear membranes is lost. Thus, cells can be separated into stages of cell death. Annexin V+/7-AAD- cells are proapoptotic/apoptotic and Annexin V+/7-AAD+ cells have completed the apoptotic process [42]. Ten thousand events were acquired on a FACSort (Becton Dickinson) flow cytometer and analyzed with CellQuest (Becton Dickinson) software for each sample of CD34+ cells.

CFC Assays
Cultured human cells were evaluated for CFC-Mix, CFC-granulocyte-macrophage (CFC-GM) and BFU-E by plating 1,000 cultured CD34+ cells from culture condition (A) or the same volume of cultured cells of culture conditions (B-F above) in triplicate in 1 ml Marrow-Gro (generously provided by Quality Biologicals) methylcellulose medium supplemented with recombinant human KL (50 ng/ml), IL-3 (10 ng/ml), GM-CSF (10 ng/ml), and erythropoietin (5 U/ml). Colonies were scored after 2 weeks. CFC assays were also performed on 105 bone marrow (BM) cells from transplanted NOD/SCID mice using these same conditions, which are selective for growth of human (not mouse) CFC [39,41,43–45].

Transplantation of Human Cells into NOD/SCID Mice for Quantitation of SEP
NOD/SCID mice were bred and maintained under pathogen-free conditions, as approved by the Animal Care Committee of the Johns Hopkins Medical Institutions [39,41,44,45]. Four sublethally irradiated (300 cGy using a 137Cs {gamma}-irradiator) 6- to 8-week-old mice were transplanted by tail vein injection with 3 x 105 cells from each culture condition evaluated. Mice were sacrificed 8-12 weeks after transplantation, and BM cells were flushed from the removed femurs and tibias. Cells were counted and their viability was determined. Human cells in the BM of human-mouse chimeras were enumerated and cell lineages determined by three-color flow cytometry of cells immunostained with murine mAb against human leukocyte differentiation antigens, as described previously [39,41,44,45].

Western Blot Analysis to Detect FLIP
Cells were washed with ice-cold isotonic phosphate buffered saline (pH 7.4, 0.05M phosphate) and then lysed at 100°C for 5 minutes in 2x Laemmli sample buffer. After boiling for 5 minutes, lysates were spun (14,000 rpm, 5 minutes, room temperature), and supernatants collected. Fifty ml of each cell lysate supernatant were electrophoresed on a 12% SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (Millipore Co.; Bedford, MA; http://www.millipore.com). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (1M Tris, pH 7.6; 100 mM NaCl) containing 0.1% polyoxyethylene-sorbitan monolaurate (Sigma). Next, the blots were probed with the appropriate dilution of the rat anti-human FLIP mAb (1 hour, room temperature) and revealed with HRP-conjugated goat anti-rat antibody. After six 1-hour washes, the blots were developed using the Supersignal® West Pico chemiluminescence method (Pierce Chemical Co.; Rockford, IL; http://www.piercenet.com) according to the manufacturer's protocol.

Reverse Transcription and Real-Time Polymerase Chain Reaction (PCR) Quantitation of Caspase 8, Fas, and FLIP mRNA Expression in CD34+ Cells
Total RNA was isolated from cells with either Trizol reagent (Life Technologies; Rockville, MD; http://www.lifetech.com) or RNeasy Mini-Kit (Qiagen; Santa Clarita, CA; http://www.qiagen.com). RNA concentration was assessed with Ribo-green Reagent (Molecular Probes; Eugene, OR; http://www.probes.com). One µg total RNA was reverse transcribed with oligo-dT primer (Life Technologies) and SuperScript II enzyme (Life Technologies). Quantitative real-time PCR was then performed on an iCycler IQ System (Bio-Rad; Hercules, CA; http://www.bio-rad.com) with 1 ng reverse transcribed total RNA (equivalent to 103 cells) per reaction. PCR reactions were carried out with Platinum Taq Polymerase (Life Technologies) with the addition of SYBR-green I (Molecular Probes) at a 1:50,000 dilution. ß-actin primers were purchased from Continental Lab Products (San Diego, CA; http://www.clpdirect.com); 5'-TGACGGGGTCACCCACACTGTGCC-3' and 5'-TAGAAGCATTTGCGGTGGACGATG-3'. The following sense and antisense primers, purchased from Operon Technologies (Alameda, CA; http://www.operon.com), are specific for separate exons within each target gene. Each primer pair spans an intervening intron within its respective target gene to preclude the detection of contaminating genomic DNA:

Caspase 8: 5'-ATGCAGGGGCTTTGACCACGAC-3' and

5'-TCCCCCTGACAAGCCTGAATAAAA-3';

Fas: 5'-TGCACCCGGACCCAGAATACCA-3' and

5'-AAGAAGAAGACAAAGCCACCCCAAGTTAGA-3';

FLIP: 5'-TGATGGCAGAGATTGGTGAGGATTTG-3' and

5'-CTTTTGGATTGCTGCTTGGAGAACATT-3'.

Thermocycles were one cycle at 95°C (2 minutes); 40 cycles at 95°C (15 seconds), 60°C (20 seconds), 72°C (30 seconds). PCR product was detected during the 72°C elongation step. DNA plasmids containing the respective mRNA sequences were used to create standard curves for quantification of each mRNA. Each result was normalized to the amount of ß-actin mRNA detected in that sample.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Culture of CD34+ Cells from Human CB or PBSC in Medium Containing sFasL or Agonistic Anti-Fas mAb Did Not Induce Apoptosis
CB CD34+ cells were cultured, with or without FKT, and with or without sFasL. In the 5-hour cultures, the presence of FKT had no effect on apoptosis, and the inclusion of sFasL had no significant impact on the numbers of cells that were either proapoptotic/apoptotic (Annexin V+/7-AAD-) or apoptotic/dead (Annexin V+/7-AAD+). The percentages of apoptotic cells were relatively low in the 5-hour culture, whereas cell preparations cultured for 48 hours without FKT contained approximately 50% apoptotic/dead (Annexin V+/7-AAD+) cells. Nevertheless, the presence of sFasL did not significantly increase cell death in cell preparations cultured for 48 hours in the presence or absence of FKT (Figs. 1A, 1BGo). Viability was also assessed by trypan blue dye exclusion at 5 or 48 hours of culture and was consistent with these findings (data not shown). As a control, sFasL was toxic to Jurkat human T leukemia cells or activated normal T cells from human blood or mouse spleen (data not shown).




View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Exposure of human CD34+ cells to sFasL did not induce apoptosis. Recombinant human sFasL (0, 1, or 10 ng/ml) was added to cultures of CB CD34+ cells that contained FKT (labeled FKT on the x-axis) or had no FKT (labeled 0) for 5 hours (A) or 48 hours (B), or of mobilized PBSC CD34+ cells (C = 5 hours and D = 48 hours). Cells were incubated with Annexin V and 7-AAD, and analyzed by FACS. Results (% apoptotic cells, mean ± standard error [SE]; error bars not shown in two groups of [B] with only a single data point) are shown for three experiments at the 5-hour time point and five experiments at the 48-hour time point; the total numbers of cells were not significantly different within the FKT or the no FKT groups.

 
sFasL did not result in apoptosis of PBSC CD34+ cells (Figs. 1C, 1DGo), evaluated as above, and treatment of cells with the CH11 agonistic anti-Fas mAb (instead of sFasL) gave the same results (data not shown).

Culture of CD34+ Cells in Medium Containing sFasL Did Not Inhibit Generation of CFC
CD34+ cells from CB or PBSC were cultured as above, then plated in CFC assays. There was no decrease in number of any type of CFC after exposure of CD34+ cells to sFasL for either 5 (data not shown) or 48 hours (Fig. 2AGo shows CB results; PBSC results were similar). Indeed, there were higher numbers of CFC-GM and total CFC in cultures treated with 1 ng/ml sFasL in these two experiments; however, this increase was not statistically significant.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Exposure of human CD34+ cells to sFasL or agonistic anti-Fas antibody did not decrease CFC. Recombinant human sFasL (0, 1, or 10 ng/ml in the presence or absence of FKT, as indicated by the x-axis labels, as in Figure 1Go) (A) or the agonistic anti-Fas mAb (0, 0.5, or 2.5 µg CH11 in the presence of FKT, as indicated by the x-axis labels) (B) was added to cultures of human CB CD34+ cells, incubated for 48 hours, then plated in methylcellulose medium. Resulting CFC (mean ± SE) are shown for two separate experiments. Similar results were obtained for PBSC (data not shown).

 
Similar results were obtained using the CH11 agonistic anti-Fas mAb instead of sFasL (Fig. 2BGo), except no increase in CFC was noted at the two concentrations of mAb tested.

In four additional experiments (data not shown), extended suspension cultures of CB and PBSC CD34+ cells in FKT plus or minus sFasL for 1 or 2 weeks showed no effect of sFasL on cell numbers, apoptotic cells, or CFC.

Culture of CB CD34+ Cells in Medium Containing sFasL Did Not Inhibit Engraftment in NOD/SCID Mice (SEP)
In two experiments, CB CD34+ cells, exposed to sFasL as above, were transplanted into NOD/SCID mice. Culture of CD34+ cells with sFasL prior to transplant did not consistently decrease the numbers of human cells found in the BM of mice 8-10 weeks later (Fig. 3AGo). In addition, no differences between sFasL-exposed and control groups were detected in percentages of human leukocyte types determined using mAb against CD19, CD16/56, CD13/33, CD41a, and CD71 [39]. Finally, the numbers or types of human CFC generated by BM cells from the transplanted mice were not significantly lower in sFasL-treated versus control groups (Fig. 3BGo). There were trends to higher percentages of engrafted human cells and CFC generated from the cultures treated with 1 ng/ml sFasL (compare above results for CFC).




View larger version (26K):
[in this window]
[in a new window]
  		Figure 3. Exposure of human CD34+ cells to sFasL did not inhibit SEP. A) CB CD34+ cells were exposed to sFasL as above for 48 hours prior to transplant into NOD/SCID mice. Eight to 10 weeks later, mice were sacrificed and their marrow cells analyzed by FACS for the presence of CD45+ human cells. Each point represents the percentage of human cells present in the marrow of one mouse. Two separate experiments were conducted. X-axis labels as in Figures 1, 2GoGo. B) At the time of sacrifice of the NOD/SCID mice transplanted with control or sFasL-exposed CD34+ cells, marrow cells were isolated, counted, and plated in CFC assays. Values as in Figure 2Go. X-axis labels as in Figures 1–2GoGo.

 
CD34+ Cells Expressed Caspase 8, Fas, and FLIP mRNA With a Low Ratio of Caspase 8 to FLIP
Total RNA was isolated from Jurkat, U266, PBSC CD34+, and CB CD34+ cells. After poly-A mRNA reverse transcription, the number of copies of Caspase 8, Fas, and FLIP message (Fig. 4Go) was quantified from 1 ng samples of total RNA (approximately 103 cells). Fas expression (Fig. 4BGo) varied among the different cell types with Jurkat cells (5.0 x 105 copies) expressing ~10 times as much Fas mRNA as U266 cells (7.4 x 104 copies). PBSC CD34+ (2.0 x 104) and CB CD34+ (6.8 x 103) cells expressed Fas mRNA, but at levels much lower than in the control cell lines. PBSC CD34+ cells (1.2 x 105 copies) and CB CD34+ cells (7.3 x 104 copies) expressed levels of Caspase 8 mRNA (Fig. 4AGo) comparable with that of U266 cells (4.1 x 105 copies), but ~10-fold lower than in Jurkat cells (4.2 x 106 copies). U266 (4.3 x 104 copies), PBSC CD34+ (5.3 x 104 copies), and CB CD34+ (1.4 x 104 copies) cells expressed FLIP mRNA (Fig. 4CGo) at comparable, high levels, ~100 times higher than in Jurkat cells (7.9 x 102 copies). The ratio of Caspase 8:FLIP (Fig. 4DGo) was 5,255:1 for Jurkat cells, 9.6:1 for U266 cells, 2.2:1 for PBSC CD34+ cells, and 5.3:1 for CB CD34+ cells.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Expression of Caspase 8, Fas, and FLIP mRNA in human CD34+ cells. Reverse transcribed mRNA was analyzed by real-time PCR to quantify expression of Caspase 8 (A), Fas (B), and FLIP (C) mRNA within CB CD34+, PBSC CD34+, Jurkat T cells (known to be sensitive to Fas-induced apoptosis), and U266 human myeloma cells (known to express FLIP). Expression of each mRNA is shown as the number of copies in 1 ng RNA detected by real-time quantitative PCR. The ratio of Caspase 8:FLIP (D) was calculated for each cell type. Results are expressed as the log scale of copies of Caspase 8 to copies of FLIP mRNA. These results are representative of three independent experiments.

 
CD34+ Cells Expressed FLIP Protein
FLIP levels were measured in CB CD34+ and CD34- cell preparations by Western blotting. FLIP levels were as high or higher in CB CD34+ cells than in the positive control U266 human myeloma cells (known to express high levels of FLIP [46]). In contrast, CD34- cells did not express detectable FLIP, even when >10-fold more CD34- cells were analyzed (Fig. 5Go).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 5. Human CD34+ cells expressed FLIP protein. Lysates of U266 human myeloma cells (known to express high amounts of the 55 kDa isoform of FLIP) or CB CD34+ or CD34- cells were analyzed by Western blot using rabbit anti-human FLIP primary mAb and donkey anti-rabbit secondary antibody as described. A representative blot (of three experiments) of 1.2x106 CB CD34+ cells (96% purity) contained higher levels of FLIP than did that number of U266 cells or 1- or 10-fold that number of CB CD34- cells. An additional lane with 67-fold that number of CD34- cells had high background, but the FLIP band could still not be discerned (data not shown). Similar results were obtained using CD34+ cells from adult PBSC (data not shown).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
The Fas pathway is key in the regulation of a number of cellular responses. A well-defined example is the limitation of a given immune response by activation-induced cell death (AICD) of activated T lymphocytes. The Fas pathway is the major effector of AICD [47–49], and activation of T cells markedly increases their sensitivity to apoptosis upon exposure to FasL [1,50–52]. Teleologically, it would be beneficial for HSC to be protected from Fas-mediated death signals that may be present in the environment for unrelated purposes. Our results show that exposure of human neonatal CB or adult PBSC CD34+ cells to sFasL (or agonistic anti-Fas mAb) did not adversely affect either their viability/apoptosis or their hematopoietic function, by in vitro CFC and in vivo SEP assays. At doses of sFasL that were highly toxic to T cells, no significant effect was observed on CD34+ cells. The resistance of HSC to Fas-mediated apoptosis may be an important mechanism of their survival in vivo. Our findings are consistent with previous studies in which HSC were found to be resistant to engagement of the Fas pathway [27,28]. Indeed, our data include a trend to increased survival of CFC and SRC (Figs. 2, 3GoGo) after treatment with 1 ng/ml sFasL, which may also be consistent with the suggestions of two reports that sFasL may have some anti-apoptotic and even proliferative effects on CD34+ cell subsets [27,28]. Further experiments will be needed to address sFasL signaling (e.g., FLIP levels, proliferative signaling targets) in early CD34+ cells.

Prior studies of molecular mechanisms of sensitivity of HSC to Fas-mediated apoptosis have focused on whether or not HSC express Fas [7,31]. Clearly, HSC that do not express any Fas (receptor) should be resistant to Fas-mediated apoptosis. However, prior reports [27,28] found that CD34+ cell subsets expressed detectable amounts of Fas, although the early CD34+/CD38- cell subset had very low Fas levels. We extended this, not only by demonstrating Fas-resistance of in vivo engrafting human HSC, but also by demonstrating that the Fas pathway inhibitor, FLIP, was highly expressed in CD34+ cell mRNA and protein. FLIP acts as a potent competitive inhibitor of Caspase 8. At a ratio as little as one FLIP molecule to 100 Caspase 8 molecules, FLIP has been shown to effectively inhibit Fas-triggered apoptosis [23]. Jurkat T cells are known to be highly sensitive to Fas-induced apoptosis, while U266 myeloma cells are resistant. In keeping with their sensitivities, Jurkat cells expressed ~10 times as much Fas mRNA as U266 cells. In addition, Jurkat cells contained over 5,000 times as much Caspase 8 as FLIP mRNA, whereas U266 cells expressed only 10 times as much Caspase 8 as FLIP. PBSC CD34+ and CB CD34+ cells expressed Fas message, but at very low levels. At the same time, they displayed Caspase:FLIP ratios lower than even the apoptosis-resistant U266 cells. The very low, albeit still present, amount of Fas and the very high expression of FLIP may each tend to protect CD34+ cells from apoptosis. The adult CD34+ cells we studied were obtained from donors treated with pharmacologic doses of cyclophosphamide and G-CSF. We confirmed these results by testing (untreated) neonatal CB CD34+ cells. Nevertheless, additional confirming studies should be done on CD34+ cells from bone marrow of untreated adults to definitively rule out the unlikely possibility that a treatment effect might be responsible for inducing Fas resistance and FLIP expression in adult CD34+ cells.

Our finding that FLIP is involved in Fas pathway inhibition in HSC suggests further investigation of molecules that may enhance survival and function of CD34+ cells. FLIP and other potential inhibitors of cellular apoptosis (e.g., Bcl-2 and Bcl-XL) can be induced by exposure to growth factors and may contribute to resistance to sFasL in diverse cell types. Recently, it was reported [26] that CD34+ cells express high levels of survivin, a member of the IAP family. Survivin levels were upregulated by exposure of CD34+ cells to the FKT growth factors. In the future, it will be interesting to examine the effects of selected cytokines on expression/activity of Fas, FLIP, and each key member of the cellular apoptotic pathway in order to further elucidate the molecular basis for hematopoiesis.

Regulation of the Fas pathway, as well as other apoptotic mediators, may also be important in dysregulation of hematopoiesis, since failure of HSC to develop Fas sensitivity during maturation might constitute a malignant "hit" [53]. One study demonstrated that CD34+/CD38- leukemia cells were less sensitive to Fas-mediated apoptosis than CD34+/CD38- cells from normal individuals [37]. Thus, understanding the Fas pathway in HSC may explain some mechanisms of leukemogenesis and reveal potential targets for novel therapies.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Human CB and PBSC CD34+ HSC were resistant to Fas pathway agonists. High-level expression of FLIP likely provides one level of protection of CD34+ cells from Fas-mediated apoptosis. Regulation of the Fas pathway, as well as other apoptotic mediators, may be important in the normal physiology of hematopoiesis and the pathophysiology of certain hematopoietic disorders. For example, understanding the Fas pathway in HSC may explain some mechanisms of leukemogenesis and reveal potential targets for novel therapies.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
This work was supported in part by grant #P01CA70970 from the National Institutes of Health and a Translational Research Award from the Leukemia and Lymphoma Society of America.

The Johns Hopkins University holds patents on CD34 mAb and related inventions. CIC is entitled to a share of the sales royalty received by the University under licensing agreements between the University, Becton Dickinson Corporation, and Baxter HealthCare Corporation. This arrangement is being managed by the University in accordance with its conflict of interest policies.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. George JF, Sweeney SD, Kirklin JK et al. An essential role for Fas ligand in transplantation tolerance induced by donor bone marrow. Nat Med 1998;4:333–335.[CrossRef][Medline]

  2. Green DR, Ware CF. Fas-ligand: privilege and peril. Proc Natl Acad Sci USA 1997;94:5986–5990.[Free Full Text]

  3. Takahashi T, Tanaka M, Inazawa J et al. Human Fas ligand: gene structure, chromosomal location and species specificity. Int Immunol 1994;6:1567–1574.[Abstract/Free Full Text]

  4. Ehl S, Hoffmann-Rohrer U, Nagata S et al. Different susceptibility of cytotoxic T cells to CD95 (Fas/Apo-1) ligand-mediated cell death after activation in vitro versus in vivo. J Immunol 1996;156:2357–2360.[Abstract]

  5. Niederkorn JY. The immune privilege of corneal allografts. Transplantation 1999;67:1503–1508.[CrossRef][Medline]

  6. O'Flaherty E, Wong WK, Pettit SJ et al. Regulation of T-cell apoptosis: a mixed lymphocyte reaction model. Immunology 2000;100:289–299.[CrossRef][Medline]

  7. Sato T, Selleri C, Anderson S et al. Expression and modulation of cellular receptors for interferon-gamma, tumour necrosis factor, and Fas on human bone marrow CD34+ cells. Br J Haematol 1997;97:356–365.[CrossRef][Medline]

  8. Griffith TS, Brunner T, Fletcher SM et al. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995;270:1189–1192.[Abstract/Free Full Text]

  9. Griffith TS, Yu X, Herndon JM et al. CD95-induced apoptosis of lymphocytes in an immune privileged site induces immunological tolerance. Immunity 1996;5:7–16.[CrossRef][Medline]

  10. Schneider E, Moreau G, Arnould A et al. Increased fetal and extramedullary hematopoiesis in Fas-deficient C57BL/6-lpr/lpr mice. Blood 1999;94:2613–2621.[Abstract/Free Full Text]

  11. Duke RC, Newell E, Schleicher M et al. Transplantation of cells and tissues expressing Fas ligand. Transplant Proc 1999;31:1479–1481.[CrossRef][Medline]

  12. Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449–1456.[Abstract/Free Full Text]

  13. Iwama H, Akutsu H, Kuretake S et al. Serum concentrations of soluble Fas antigen and soluble Fas ligand in mother and newborn. Arch Gynecol Obstet 2000;263:108–110.[CrossRef][Medline]

  14. Tanaka M, Itai T, Adachi M et al. Downregulation of Fas ligand by shedding. Nat Med 1998;4:31–36.[CrossRef][Medline]

  15. Schneider P, Holler N, Bodmer JL et al. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity. J Exp Med 1998;187:1205–1213.[Abstract/Free Full Text]

  16. Hohlbaum AM, Moe S, Marshak-Rothstein A. Opposing effects of transmembrane and soluble Fas ligand expression on inflammation and tumor cell survival. J Exp Med 2000;191:1209–1220.[Abstract/Free Full Text]

  17. Tanaka M, Suda T, Haze K et al. Fas ligand in human serum. Nat Med 1996;2:317–322.[CrossRef][Medline]

  18. Suda T, Okazaki T, Naito Y et al. Expression of the Fas ligand in cells of T cell lineage. J Immunol 1995;154:3806–3813.[Abstract]

  19. Nagata S. Apoptosis by death factor. Cell 1997;88:355–365.[CrossRef][Medline]

  20. Wang J, Lobito AA, Shen F et al. Inhibition of Fas-mediated apoptosis by the B cell antigen receptor through c-FLIP. Eur J Immunol 2000;30:155–163.[CrossRef][Medline]

  21. Siegel RM, Frederiksen JK, Zacharias DA et al. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 2000;288:2354–2357.[Abstract/Free Full Text]

  22. Perlman H, Pagliari LJ, Georganas C et al. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis. J Exp Med 1999;190:1679–1688.[Abstract/Free Full Text]

  23. Irmler M, Thome M, Hahne M et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997;388:190–195.[CrossRef][Medline]

  24. Scaffidi C, Schmitz I, Krammer PH et al. The role of c-FLIP in modulation of CD95-induced apoptosis. J Biol Chem 1999;274:1541–1548.[Abstract/Free Full Text]

  25. Domen J, Weissman IL. Hematopoietic stem cells need two signals to prevent apoptosis; BCL-2 can provide one of these, Kitl/c-Kit signaling the other. J Exp Med 2000;192:1707–1718.[Abstract/Free Full Text]

  26. Fukuda S, Pelus LM. Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34(+) cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis. Blood 2001;98:2091–2100.[Abstract/Free Full Text]

  27. Barcena A, Muench MO, Song KS et al. Role of CD95/Fas and its ligand in the regulation of the growth of human CD34++CD38- fetal liver cells. Exp Hematol 1999;27:1428–1439.[CrossRef][Medline]

  28. Josefsen D, Myklebust JH, Lynch DH et al. Fas ligand promotes cell survival of immature human bone marrow CD34+CD38- hematopoietic progenitor cells by suppressing apoptosis. Exp Hematol 1999;27:1451–1459.[CrossRef][Medline]

  29. Maciejewski J, Selleri C, Anderson S et al. Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood 1995;85:3183–3190.[Abstract/Free Full Text]

  30. Nagafuji K, Shibuya T, Harada M et al. Functional expression of Fas antigen (CD95) on hematopoietic progenitor cells. Blood 1995;86:883–889.[Abstract/Free Full Text]

  31. Maciejewski JP, Selleri C, Sato T et al. Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia. Br J Haematol 1995;91:245–252.[Medline]

  32. Young NS. Immune pathophysiology of acquired aplastic anemia. Eur J Haematol Suppl 1996;60:55–59.[Medline]

  33. Gupta P, Niehans GA, LeRoy SC et al. Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival. Leukemia 1999;13:44–53.[CrossRef][Medline]

  34. Deeg HJ, Beckham C, Loken MR et al. Negative regulators of hemopoiesis and stroma function in patients with myelodysplastic syndrome. Leuk Lymphoma 2000;37:405–414.[Medline]

  35. Shetty V, Hussaini S, Broady-Robinson L et al. Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates. Blood 2000;96:1388–1392.[Abstract/Free Full Text]

  36. Lee JW, Gersuk GM, Kiener PA et al. HLA-DR-triggered inhibition of hemopoiesis involves Fas/Fas ligand interactions and is prevented by c-kit ligand. J Immunol 1997;159:3211–3219.[Abstract]

  37. Costello RT, Mallet F, Gaugler B et al. Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities. Cancer Res 2000;60:4403–4411.[Abstract/Free Full Text]

  38. Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995;92:10119–10122.[Abstract/Free Full Text]

  39. Gao Z, Fackler MJ, Leung W et al. Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34- cell preparations. Exp Hematol 2001;29:910–921.[CrossRef][Medline]

  40. Leung W, Ramirez M, Civin CI. Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood. Biol Blood Marrow Transplant 1999;5:69–76.[CrossRef][Medline]

  41. Novelli EM, Cheng L, Yang Y et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 1999;10:2927–2940.[CrossRef][Medline]

  42. Terstappen LW, Shah VO, Conrad MP et al. Discriminating between damaged and intact cells in fixed flow cytometric samples. Cytometry 1988;9:477–484.[CrossRef][Medline]

  43. Carow CE, Harrington MA, Broxmeyer HE. Detection of human myeloid progenitor cells in a murine background. Exp Hematol 1993;21:66–69.[Medline]

  44. Leung W, Ramirez M, Novelli EM et al. In vivo engraftment potential of clinical hematopoietic grafts. J Investig Med 1998;46:303–311.[Medline]

  45. Ramirez M, Rottman GA, Shultz LD et al. Mature human hematopoietic cells in donor bone marrow complicate interpretation of stem/progenitor cell assays in xenogeneic hematopoietic chimeras. Exp Hematol 1998;26:332–344.[Medline]

  46. Kim DK, Cho ES, Yoo JH et al. FLIP is constitutively hyperexpressed in Fas-resistant U266 myeloma cells, but is not induced by IL-6 in Fas-sensitive RPM18226 cells. Mol Cells 2000;10:552–556.[Medline]

  47. Kamradt T, Mitchison NA. Tolerance and autoimmunity. N Engl J Med 2001;344:655–664.[Free Full Text]

  48. Restifo NP. Not so Fas: re-evaluating the mechanisms of immune privilege and tumor escape. Nat Med 2000;6:493–495.[CrossRef][Medline]

  49. Wahlsten JL, Gitchell HL, Chan CC et al. Fas and fas ligand expressed on cells of the immune system, not on the target tissue, control induction of experimental autoimmune uveitis. J Immunol 2000;165:5480–5486.[Abstract/Free Full Text]

  50. Eischen CM, Williams BL, Zhang W et al. ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis. J Immunol 1997;159:1135–1139.[Abstract]

  51. Ju ST, Panka DJ, Cui H et al. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 1995;373:444–448.[CrossRef][Medline]

  52. Griffith TS, Ferguson TA. The role of FasL-induced apoptosis in immune privilege. Immunol Today 1997;18:240–244.[CrossRef][Medline]

  53. Straus SE, Jaffe ES, Puck JM et al. The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis. Blood 2001;98:194–200.[Abstract/Free Full Text]

Received October 31, 2001; accepted for publication December 19, 2001.



This article has been cited by other articles:


Home page
 J. Immunol.Home page
F. Rezzoug, Y. Huang, M. K. Tanner, M. Wysoczynski, C. L. Schanie, P. M. Chilton, M. Z. Ratajczak, I. J. Fugier-Vivier, and S. T. Ildstad
TNF-{alpha} Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function
J. Immunol., January 1, 2008; 180(1): 49 - 57.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
M. Pearl-Yafe, J. Stein, E. S. Yolcu, D. L. Farkas, H. Shirwan, I. Yaniv, and N. Askenasy
Fas Transduces Dual Apoptotic and Trophic Signals in Hematopoietic Progenitors
Stem Cells, December 1, 2007; 25(12): 3194 - 3203.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Askenasy, E. S. Yolcu, I. Yaniv, and H. Shirwan
Induction of tolerance using Fas ligand: a double-edged immunomodulator
Blood, February 15, 2005; 105(4): 1396 - 1404.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Yada, N. Takamura, K. Inagaki-Ohara, M. K. O'Leary, C. Wasem, T. Brunner, D. R. Green, T. Lin, and M. J. Pinkoski
The Role of p53 and Fas in a Model of Acute Murine Graft-versus-Host Disease
J. Immunol., February 1, 2005; 174(3): 1291 - 1297.
[Abstract] [Full Text] [PDF]

Home page
 LupusHome page
M Tiefenthaler, N Bacher, H Linert, O Muhlmann, S Hofer, N Sepp, A Amberger, F Geisen, G Obermoser, and G Konwalinka
Apoptosis of CD34 cells after incubation with sera of leukopenic patients with systemic lupus erythematosus
Lupus, June 1, 2003; 12(6): 471 - 478.
[Abstract] [PDF]

Home page
 BloodHome page
K. A. Whartenby, E. E. Straley, H. Kim, F. Racke, V. Tanavde, K. S. Gorski, L. Cheng, D. M. Pardoll, and C. I. Civin
Transduction of donor hematopoietic stem-progenitor cells with Fas ligand enhanced short-term engraftment in a murine model of allogeneic bone marrow transplantation
Blood, October 16, 2002; 100(9): 3147 - 3154.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by Civin, C. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by Civin, C. I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.peprotech.com/