The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 180, 49-57
Copyright © 2008 by The American Association of Immunologists, Inc.
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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rezzoug, F.
Right arrow Articles by Ildstad, S. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rezzoug, F.
Right arrow Articles by Ildstad, S. T.

TNF-{alpha} Is Critical to Facilitate Hemopoietic Stem Cell Engraftment and Function1

Francine Rezzoug*, Yiming Huang*, Michael K. Tanner*, Marcin Wysoczynski{dagger}, Carrie L. Schanie*, Paula M. Chilton*, Mariusz Z. Ratajczak{dagger}, Isabelle J. Fugier-Vivier* and Suzanne T. Ildstad2,*

* Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202; and {dagger} Stem Cell Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR– graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-{alpha}, and FC from TNF-{alpha}-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-{alpha} in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-{alpha} is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-{alpha} in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bone marrow (BM)3 transplantation (BMT) has emerged as a promising therapeutic approach to treating numerous blood and immune-based disorders and inducing tolerance to transplanted organs (1, 2). However, the widespread use of BMT is limited by graft-vs-host disease and a requirement for closely matched donors. We previously reported that CD8+/TCR– graft facilitating cells (FC) significantly enhance engraftment of purified hemopoietic stem cells (HSC) in allogeneic and syngeneic recipients without causing graft-vs-host disease (3, 4). The majority of FC phenotypically resemble precursor plasmacytoid dendritic cells (p-preDC) (5). Removal of p-preDC FC completely abrogates facilitation. However, p-preDC FC do not replace FC total in facilitative function. Matching at MHC class I K between FC and HSC is critical to FC function (6), suggesting that cell:cell interactions between FC and HSC may be important to FC function. In the present studies, we evaluated the mechanism of FC function on HSC in vivo and in vitro. We found a critical requirement for TNF-{alpha} in the FC effects on HSC that require direct cellular contact between FC and HSC. Understanding the mechanism by which FC regulate HSC function will allow new approaches to harness the full potential of stem cell-based therapies.

The hemopoietic microenvironment plays a major role in HSC regulation, both directly through cell:cell interactions and indirectly through production of cytokines (7, 8). Many cytokines and growth factors have been shown to regulate HSC survival (9, 10, 11), homing (12, 13), and proliferation (14, 15). Among them, TNF-{alpha} is one of the most potent (16, 17). TNF-{alpha} has been demonstrated to play a pivotal role in regulating HSC proliferation directly and via stimulation of growth factor production or up-regulation of cytokine receptors (13, 18, 19). TNF-{alpha} acts as a bifunctional regulator for HSC, inducing proliferation of the more primitive subset of progenitors while simultaneously inducing a differentiation block downstream in response to hemopoietic stress and increased demand for mature blood cells (19). Until now, the cells within the hemopoietic microenvironment responsible for the regulatory effect of TNF-{alpha} on HSC have not been defined.

In the present studies, we investigated the mechanism of FC function. We found that FC significantly increase HSC survival and maintain pluripotency of HSC in vitro. As FC are composed primarily of p-preDC and produce high levels of TNF-{alpha} under TLR9 stimulation (5), we explored whether TNF-{alpha} is involved in FC function. In contrast with CpG oligodeoxynucleotide (ODN) stimulation, where high levels of TNF-{alpha} were produced by FC, direct contact between FC and HSC induced FC to produce low levels of TNF-{alpha}. We found that TNF-{alpha} is critical to FC function in syngeneic and allogeneic recipients in vivo as well as in vitro. As a novel engraftment-enhancing cell with potent biologic effects in vivo, FC could have a profound effect on the safe use of stem cell-mediated therapies for autoimmune diseases, to induce tolerance for solid organ allografts, and in expanding criteria for alternative donors for BMT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Four-week-old C57BL/6J (B6; H-2b) and B10.BR (BR; H-2k) mice (The Jackson Laboratory), TNF-{alpha}–/– mice (TNF KO; H-2b B6 background), and TNF-{alpha} receptor-deficient mice (TNFR–/–, H-2b B6 background; a gift from Dr. David H. Lynch, Immunex Corporation, Seattle, WA) were bred at the Institute for Cellular Therapeutics. Animals were cared for according to National Institutes of Health guidelines.

Cell preparation

Cells were isolated from BM by multiparameter, live sterile cell sorting (FACSVantage SE; BD Biosciences), as previously described (3, 5). HSC were sorted for c-Kit+/Sca-1+/Lin– (KSL) cells, FC for CD8+/TCR{alpha}β–/TCR{gamma}{delta}–, T cells for CD8+/TCR{alpha}β+/TCR{gamma}{delta}+, p-preDC for CD11c+/B220+/CD11b–, and p-preDC FC for CD8+/TCR–/B220+/CD11c+ expression. All Abs were purchased from BD Biosciences/BD Pharmingen.

Transplantation

Mice were conditioned with 950 cGy total body irradiation (gamma cell 40; Nordion) and transplanted by tail vein injection 6 h after irradiation. Donor chimerism testing was performed as previously described (4).

Cell culture

Sorted cells were cultured for 18 or 40 h at 15,000 KSL cells alone or plus 30,000 FC from B6 or from TNF-{alpha}–/– mice or p-preDC, p-preDC FC, or T cells in long-term culture medium (LTCM; IMEM (Invitrogen Life Technologies) with 20% horse serum (Invitrogen Life Technologies), 10–6 M hydrocortisone (Sigma-Aldrich), 10–5 M 2-ME (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen Life Technologies), 2 mM L-glutamine (Invitrogen Life Technologies), and 25 mM NaHCO3 (Sigma-Aldrich)) in a 96-well plate and incubated at 37°C. After culture, 100 µl of supernatant (SN) was removed and frozen for further analysis, cells were resuspended and used in the colony-forming cell assay (CFC) assay. In some experiments, KSL cells were cultured with 10 ng/ml of TNF-{alpha} (Genzyme) or with a 1/10 dilution of SN from 18 h culture of FC plus KSL cells. In experiments with anti-TNF-{alpha}, FC were preincubated for 1 h with 100 ng/ml anti-TNF-{alpha} mAb or isotype control (BD Pharmingen), and then cocultured as described with KSL cells. In some experiments, KSL cells were cultured in the lower chamber of 96-well plates with a transwell insert (BD Biosciences) and FC in the upper chamber or lower chamber (control).

Cobblestone area-forming cell (CAFC) and long-term culture-initiating cell (LTC-IC) assays

CAFC assays were performed as described (20, 21). Limiting dilutions of KSL cells were added to a pre-established FBMD1 stromal layer (22) (provided by Dr. G. Van Zant, University of Kentucky, Lexington, KY) in the absence or presence of 500 FC/well in LTCM and incubated at 33°C. Half of the medium was changed weekly. The CAFC were evaluated at days 10 and 28. To evaluate LTC-IC, the medium was removed on day 35, and 100 µl of methylcellulose-containing mouse growth factors (MethoCult GF M3434; StemCell Technologies) we added to the culture. The plates were incubated at 37°C for 14 more days, and positive wells were enumerated.

Colony-forming cell assay

CFC assay was performed on freshly sorted cells or after 18 or 40 h of culture. KSL cells were suspended at 100 cells/ml in methylcellulose and cultured in duplicate at 37°C for 14 days. Colonies containing >50 cells were scored.

SN preparation and cytokine evaluation

FC or T cells (100,000) were cultured alone, with KSL cells (30,000), or with 1 µM TLR9 ligand CpG ODN 1668 (TCCATGACGTTCCGATGCT; Invitrogen Life Technologies Custom Primers). SN were collected 18 h later and stored at –20°C. Evaluation of cytokines present in SN was performed by Linco Diagnostic using a LINCOplex Multiplex Immunoassay for mouse cytokines: MIP-1{alpha}, GM-CSF, MCP, KC, RANTES, IFN-{gamma}, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-{alpha}, IL-9, and IL-13 (Linco Diagnostic).

Apoptosis assay

A total of 15,000 KSL cells were incubated alone or in the presence of 45,000 B6 FC or TNF-{alpha}–/– FC in a 96-well round-bottom plate for 18 or 40 h in RPMI 1640 (Invitrogen Life Technologies) containing 2% FBS (HyClone), 10–5 M 2-ME, L-glutamine, penicillin/streptomycin. After incubation, Fc{gamma}Rs were blocked with anti-CD16/CD32 Ab; cells were stained with c-Kit-allophycocyanin mAb and Annexin V-FITC (BD Pharmingen) for 20 min. Cell death was measured using 0.25 µg/ml 7-aminoactinomycin D (7-AAD; Molecular Probes) by flow cytometry. Lymphoid-gated c-Kit+ cells were separated into three categories based on annexin V and 7-AAD staining patterns.

Real-time RT-PCR

KSL cells were incubated for 16 or 22 h with or without TNF-{alpha} (10 ng/ml) as for the apoptotic assay. After incubation, Fc{gamma}Rs were blocked, cells were stained with c-Kit-allophycocyanin, and resorted for c-Kit+ (KSL cells) and c-Kit– cells (FC). Total mRNA was isolated with the RNeasy Mini kit (Qiagen) and reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems). Detection of Bcl-xL, Bax, FLIP, Bcl-2, Bcl-3, p53, TNF-{alpha}, and β2-microglobulin (housekeeping gene as internal control) mRNA levels was performed by real-time RT-PCR using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Each 25-µl reaction mixture contained 12.5 µl of SYBR Green PCR Master Mix, 10 ng of cDNA template, and primer mRNA. Primers mRNA were designed with the Primer Express software (Table I). Relative quantitation of Bcl-xL, Bax, FLIP, Bcl-2, Bcl-3, p53, and TNF-{alpha} mRNA expression was calculated with the comparative threshold cycle method as described elsewhere (23).


View this table:
[in this window]
[in a new window]

  		Table I. Primer sequences used for real-time RT-PCR assay

 
CFSE labeling for HSC proliferation

KSL cells were washed using 1x HBSS and labeled with 0.5 µM CFSE (Molecular Probes) for 5 min at 37°C. The reaction was stopped and cells were washed in LTCM. KSL cells were cultured in the absence or presence FC or T cells for 3–5 days at 37°C. After culture, cells were labeled as for the apoptosis analysis, then analyzed by flow cytometry for CFSE+/c-Kit+ cells. Data were analyzed using the Mod Fit program; results give the percent of proliferation as 100% minus the percent of parents, for at least four experiments at each time point.

Statistical analysis

Statistical analyses were performed using the Student t test. To calculate significance for CFC assay and cell survival/apoptosis assays, a t test for pairwise comparison was performed. Data were considered significantly different when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
FC significantly enhance HSC function in vitro

We first evaluated the effect of FC on HSC using in vitro assays. The term KSL cell (c-Kit+/Sca-1+/Lin– cell) will be used to refer to HSC sorted by this phenotype. KSL cells are a heterogeneous population comprised of short-term and long-term repopulating cells as well as more committed progenitors. The CAFC assay allows detection of committed (day 10 CAFC) and more primitive progenitors (day 28 CAFC) in the KSL cell population (20). KSL cells were cultured for 5 wk in the presence or absence of FC. The frequency of committed and more primitive progenitors was significantly increased when KSL cells were cultured in the presence of FC compared with KSL cells cultured alone (Fig. 1, A and B; p < 0.05). The frequency of LTC-IC, containing long-term repopulating cells, was also significantly increased when KSL cells were cocultured with FC (Fig. 1C; p < 0.05). Although FC alone did not generate colonies, the wells with FC maintained a better stromal layer, suggesting that FC contribute to the BM microenvironment (data not shown).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 1. FC enhance HSC function in vitro. A and B, CAFC assay. A, Early CAFC enumerated at day 10 reflective of committed progenitors. KSL cells were cultured for 5 wk at a limiting dilution on a stromal layer in the presence or absence of FC. B, Late CAFC reflecting more primitive HSC populations evaluated at day 28. These figures represent data from four different experiments. C, LTC-IC assay performed at day 35 of CAFC for the average of the different experiments. D, CFC assay performed on freshly sorted KSL cells mixed with FC or T cells. E, Analysis of colony types in CFC assay. Results are averaged from six experiments. For all figures, each triangle ({blacktriangleup}) represents one individual sample. The dashed lines (----) link samples from the same experiment. Results are expressed as frequency of colonies per 1000 KSL cells (*, p < 0.05).

 
We then evaluated the role of FC in promoting KSL cell differentiation in methylcellulose-containing growth factors, the CFC assay. In this assay, monolineage colony formation reflects the differentiation of a progenitor committed to one lineage, while multilineage colony production reflects differentiation of a more primitive multipotent progenitor. Overall, the number of colonies generated was significantly increased when FC were cocultured with KSL cells (Fig. 1D; p < 0.05). The number of multilineage colonies was also significantly increased when KSL cells were cocultured with FC compared with KSL cells alone (Fig. 1E, left; p < 0,05), confirming that FC promote a higher frequency of early progenitors. FC alone did not generate colonies (Fig. 1D). T cells used as a negative control did not increase KSL cell clonogenicity, nor did they generate colonies (Fig. 1D).

We recently identified p-preDC as the critical effector cell in the FC population for allogeneic HSC engraftment (5). Removal of p-preDC FC completely abrogates FC function. However, p-preDC FC facilitate HSC engraftment only half as efficiently as FC total. We therefore evaluated the effect of p-preDC FC, as well as p-preDC, on KSL cell clonogenicity and their contribution to FC function in vivo. There was not a significant increase in the number of colonies generated by KSL cells when they were cocultured p-preDC FC (Fig. 2A). In vivo, p-preDC FC enhanced engraftment of suboptimal numbers of HSC in syngeneic recipients, but significantly less effectively than FC total (Fig. 2B; p < 0.05). Taken together, these data suggest that p-preDC FC do not replace FC total for the full biologic effect, suggesting the need for a collaborative cell within the FC total population.


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 2. FC enhance HSC function in vitro and in vivo more efficiently than p-preDC or p-preDC FC. A, CFC assay for freshly sorted KSL cells mixed with p-preDC FC or p-pre-DC sorted from BM. Data represent four different experiments. Each triangle ({blacktriangleup}) represents one individual sample. The dashed lines (----) link samples from the same experiment; results are expressed as CFC frequency per 1000 KSL cells. B, Syngeneic model for facilitation: Kaplan-Meier survival for B6 recipients of syngeneic 500 KSL cells (----), 500 KSL cells and 30,000 FC (— · · —), 500 KSL cells and 30,000 p-preDC FC (—), irradiation control (.......); *, p < 0.05; **, p < 0.003; ***, p < 0.15.

 
FC promote HSC survival in vitro

Because preventing cell death increases homing and engraftment (24, 25, 26), we evaluated whether FC protect HSC from undergoing apoptosis. KSL cells were cultured with or without FC. FC significantly prevented apoptosis of KSL cells in culture (p < 0.05). The percentage of live cells was significantly increased when KSL cells were incubated with FC for 18 h (Fig. 3A) and 40 h (Fig. 3B), while the percentage of apoptotic cells was significantly reduced (data not shown). To determine which regulatory genes were induced in FC and HSC after coculture, KSL cells were resorted after coculture with FC and transcripts for antiapoptotic and proapoptotic molecules analyzed by real-time RT-PCR. Bcl-3 transcripts were significantly increased in KSL cells cocultured with FC (Fig. 3C, upper panel). No difference in regulatory molecule transcription was observed in the FC after coculture with KSL cells (Fig. 3C, lower panel).


Figure 3
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 3. FC increase the survival and clonogenicity of KSL cells. Evaluation of percent of live KSL cells by flow cytometry using annexin V and 7-AAD labeling after preincubation with FC or T cells for 18 h (A) or 40 h (B). Results represent four different experiments (*, p < 0.05). C, KSL cells and FC were cocultured for 16 h, then resorted, and real-time RT-PCR was performed for anti- and proapoptotic regulators. Results represent the average of three experiments. D–F, CFC assay performed after 18 h (D and F) or 40 h (E). KSL cells were preincubated with FC or T cells (D and E) or with p-preDC FC or p-preDC (F). Data represent at least four different experiments. Each triangle ({blacktriangleup}) or diamond ({diamondsuit}) represents one individual sample. The dashed lines (----) link samples from the same experiment. Results are expressed as frequency of colonies per 1000 KSL cells (*, p < 0.05).

 
To assess the function of the surviving HSC, we tested them in the CFC assay. KSL cells coincubated with FC for 18 h, then cultured in methylcellulose, generated significantly more colonies and a greater number of multilineage colonies (Figs. 3D and 1E, right) compared with KSL cells cultured alone (Fig. 3D). After 40 h, KSL cells cultured alone were no longer able to generate colonies. Strikingly, there was a 30-fold increase in the number of CFC for KSL cells cocultured with FC (Fig. 3E). The p-preDC FC and p-preDC populations did not mediate a similar effect on promoting KSL cell clonogenicity compared with FC total on freshly sorted cells (Fig. 3F), after 18 or 40 h preincubation (data not shown), corroborating our in vivo data that the p-preDC FC and p-preDC do not replace the FC effect on HSC (5).

HSC stimulate FC to produce TNF-{alpha}

FC are comprised primarily of p-preDC FC and produce high levels of TNF-{alpha} under TLR9 stimulation (27). We therefore evaluated whether coculture of HSC with FC induced production of cytokines by FC. KSL cells were cocultured with FC for 18 h, then the SNs were collected and tested on KSL cells in the apoptosis and CFC assays. We found that FC:KSL cell SNs significantly increased KSL cell clonogenicity at levels equivalent to FC (Fig. 4A). However, the SN did not replace the antiapoptotic effect of FC on KSL cells, suggesting a requirement for cell:cell contact in mediating the antiapoptotic effect (Fig. 4B). When KSL cells were separated from FC in culture with a 1-µm pore-size membrane, FC did not enhance KSL cell clonogenicity (Fig. 4C), confirming that direct contact between FC and HSC is required to produce the complete biologic effect.


Figure 4
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 4. SNs from FC cocultured with KSL cells promote HSC clonogenicity but not survival. SN preparation and evaluation of cytokines and chemokines. FC or T cells (100,000) were cultured alone or in the presence of KSL cells 30,000), or with 1 µM TLR9 ligand CpG ODN 1668. Cell-free SN were collected 18 h later. Sorted KSL cells were cultured alone, with FC, or in the presence of a 1/10 dilution of the collected SNs. A, CFC assay after 18 h of preincubation. Results are given as number of CFC for 1,000 KSL cells. (*, p < 0.05). B, Apoptosis assay after 18 h culture; results are given in percent of live KSL cells. C, KSL cells were preincubated for 18 h in direct contact with FC or separated by a 1-µm pore membrane and evaluated in the CFC assay. D, TNF-{alpha} mRNA is up-regulated in FC after16 h culture with KSL cells but not after 22 h. Results are a mean ± SD of at least three experiments. E, Cytokine production by FC after 18 h of culture with KSL cells. CpG ODN-treated FC, KSL cells alone, and unmanipulated FC served as controls. Results are given in picograms per milliliter, for an average of three samples. F, CFC after 18 h culture of KSL cells in the presence of FC, KSL cells with SN alone, or FC with or without anti-TNF-{alpha} neutralizing Ab. Results are given as number of CFC for 1,000 KSL cells. Each triangle ({blacktriangleup}) represents one individual sample. The dashed lines (----) link samples from the same experiment.

 
The fact that SN generated from FC cocultured with KSL cells enhanced KSL cell clonogenicity prompted us to explore which cytokines are induced in FC by contact with KSL cells. We previously determined that FC produce high levels of TNF-{alpha} after stimulation with CpG ODN (5). We therefore evaluated whether KSL cells would similarly induce FC to produce TNF-{alpha}. FC were cocultured with KSL cells, resorted, and analyzed for TNF-{alpha} transcript. When FC were cocultured with KSL cells for 16 h, there was a 3-fold increase in TNF-{alpha} mRNA compared with FC incubated alone (Fig. 4D). This increase was transient, as at 22 h no difference in TNF-{alpha} transcription was observed. The analysis of SN generated after FC:KSL cell coculture by cytokine array showed production of MCP, and low levels of IL-6 and IFN-{gamma}, but neither TNF-{alpha} nor the other cytokines tested were detectable (Fig. 4E). These data suggest that although FC produce TNF-{alpha} mRNA after coculture with KSL cells, the amount of TNF-{alpha} released is below the sensitivity of the assay (<3 pg/ml) or exists in membrane-bound form.

The role played by TNF-{alpha} in the SN was confirmed in CFC assays. Neutralization of TNF-{alpha} with anti-TNF-{alpha} Ab mixed with FC completely prevented the FC effect on KSL cell clonogenicity (Fig. 4F) and neutralization of TNF-{alpha} in the SN generated after coculture of FC with KSL cells also abrogated the enhanced clonogenicity effect of the SN (Fig. 4E).

FC critically require TNF-{alpha} for their facilitative function in vivo and in vitro

We used TNF-{alpha}-deficient (TNF-{alpha}–/–) mice as FC donors to compare the effect of wild-type (wt) B6 FC and TNF-{alpha}–/– FC on engraftment of B6 KSL cells in allogeneic recipients (Fig. 5A). TNF-{alpha}–/– FC were significantly impaired in facilitating KSL cell engraftment compared with B6 FC. This effect was even more profound in the syngeneic model evaluating FC-mediated engraftment of limiting numbers of B6 KSL cells in B6 recipients (Fig. 5B). These data strongly suggest a critical role for TNF-{alpha} in FC function. We hypothesize that the differences observed in the effect of TNF-{alpha}–/– FC between the syngeneic and allogeneic model could be due to the cytokines generated by alloreactivity in the allogeneic model. The accelerated graft failure observed in the group administered KSL cells plus FC from TNF-{alpha}–/– donors vs KSL cells alone may suggest that after FC bind to HSC, without early production of TNF-{alpha} by FC, the stem cell survival is significantly compromised.


Figure 5
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 5. FC from TNF-{alpha}–/– mice are functionally impaired to facilitate HSC engraftment in allogeneic and syngeneic recipients. FC were sorted from TNF-{alpha}–/– donors and transplanted with sorted KSL cells using the allogeneic and syngeneic models for facilitation. A, Engraftment in allogeneic B10.BR (H-2k) recipients transplanted with 10,000 B6 (H-2b) KSL cells alone ({blacktriangleup}) or with 30,000 FC from B6 ({diamondsuit}) or FC from TNF-{alpha}–/– (TNF-{alpha} KO; (H-2b) mice ({square}), (B) in syngeneic B6 recipients transplanted with 1,000 B6 KSL cells (•), 500 B6 KSL cells ({blacktriangleup}), 500 B6 KSL cells plus 30,000 B6 FC (or 500 B6 KSL cells plus 30,000 TNF-{alpha}–/– (TNF-{alpha} KO) FC ({blacksquare}). (*, p < 0.05). C, CFC assay for fresh KSL cells cultured in the presence of wt FC or TNF-{alpha}–/– FC. D, Percent of live KSL cells after 18 h coculture with wt FC or TNF-{alpha}–/– FC. Data represent four experiments performed in duplicate. E, CFC for KSL cells after 18 h coculture with wt FC or TNF-{alpha}–/– FC. Results are given as number of CFC for 1,000 KSL cells for at least three experiments. (*, p < 0.05 compared with KSL cells; **, p < 0.05 compared with wt FC). Each triangle ({blacktriangleup}) or diamond ({diamondsuit}) represents one individual sample. The dashed lines (----) link samples from the same experiment.

 
We also tested the function of TNF-{alpha}–/– FC on KSL cells in vitro in the clonogenicity and apoptosis assays. The function of TNF-{alpha}–/– FC was significantly impaired in both assays (Fig. 5, C and D). TNF-{alpha}–/– FC did not improve KSL cell clonogenicity in CFC assays, and the number of colonies generated was significantly lower compared with KSL cells cocultured with normal FC (Fig. 5C). The antiapoptotic effect of TNF-{alpha}–/– FC on KSL cells was also significantly impaired (Fig. 5D). Furthermore, significantly fewer colonies were generated when KSL cells were cocultured for 18 h with TNF-{alpha}–/– FC then placed in methylcellulose, compared with normal FC (Fig. 5E).

The critical requirement for TNF-{alpha} in FC function was confirmed using neutralizing anti-TNF-{alpha} Abs to block TNF-{alpha} on wt FC. The effect of FC on KSL cell clonogenicity was completely abolished when TNF-{alpha} was neutralized on FC (Figs. 4F and 6A), and KSL cell survival was significantly lower compared with untreated FC (Fig. 6B). These data strongly support a role for TNF-{alpha} produced by FC in the effect of FC on KSL cells.


Figure 6
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 6. Neutralizing TNF-{alpha} on FC abrogates the FC effects on KSL cell clonogenicity, survival, and up-regulation of the Bcl-3 transcript in KSL cells. A, CFC for KSL cells after 18 h coculture alone or in the presence of FC, or FC pretreated with anti-TNF-{alpha}-neutralizing Ab. Results are given as number of CFC for 1000 KSL cells, for four different experiments. B, Percent of live KSL cells alone or coculture with FC, or FC pretreated with anti-TNF-{alpha}-neutralizing Ab. Data represent four experiments performed in duplicate (*, p < 0.05). C, Real-time RT-PCR for resorted KSL cells after 16 h coculture with FC. D, Real-time RT-PCR for Bcl-3 transcript in KSL cells cultured for 16 h with TNF-{alpha} (10 ng/ml), or with FC or FC treated with anti-TNF-{alpha} Abs. Results are a mean ± SD of at least four different experiments. E, CFC for KSL cells after 18 h coculture with wt FC or TNF-{alpha} (10 ng/ml). Results are given as number of CFC for 1000 KSL cells for at least three experiments. F, Apoptosis assay for KSL cells after 40 h of culture with TNF-{alpha} (10 ng/ml). Results represent the percent of live KSL cells. Data represent four experiments in duplicate (*, p < 0.05). Each triangle ({blacktriangleup}) or diamond ({diamondsuit}) represents one individual sample. The dashed lines (----) link samples from the same experiment.

 
Bcl-3 up-regulation in HSC is mediated by TNF-{alpha}

We tested whether TNF-{alpha} was involved in Bcl-3 transcript up-regulation in KSL cells. KSL cells were cocultured with FC or FC preincubated with anti-TNF-{alpha} mAb, resorted, and analyzed for antiapoptotic and proapoptotic regulatory protein transcripts. Coculture of FC with KSL cells was associated with significant up-regulation of transcript for Bcl-3 in KSL cells (Figs. 3C and 6C). Blocking of TNF-{alpha} significantly impaired Bcl-3 transcription (Fig. 6C). Therefore, TNF-{alpha} is required for FC-induced up-regulation of Bcl-3 transcription in KSL cells. Moreover, incubation of KSL cells with low-dose (10 ng/ml) TNF-{alpha} resulted in a similar up-regulation of Bcl-3 transcription (Fig. 6D). However, this same dose of TNF-{alpha} did not enhance KSL cell clonogenicity (Fig. 6E), and as previously reported (18), increased KSL cell apoptosis (Fig. 6F). Taken together, these data demonstrate a role of FC on HSC function through TNF-{alpha} secretion, without the toxic and/or inflammatory effects observed with higher doses of TNF-{alpha}.

FC from wt donors partially restore engraftment of TNF-{alpha} receptor-deficient (TNFR–/–) KSL cells

To further define the role of TNF-{alpha}, we used KSL cells from TNFR–/– mice in allogeneic transplantation studies. TNFR–/– KSL cells alone were not able to engraft in allogeneic recipients. The addition of wt (B6) FC increased the short-term but not long-term engraftment of TNFR–/– KSL cells (Fig. 7A), as the survival was 20% at 200 days. In vitro, TNFR–/– KSL cells generated fewer colonies than wt KSL cells in CFC, and there was no effect of wt FC on TNFR–/– KSL cell clonogenicity of fresh cells (Fig. 7B) or after 18/40 h coincubation (data not shown). These data show that the presence of TNF-{alpha} receptors on KSL cells is not critical for early facilitation to occur in vivo, but is necessary to preserve clonogenicity and the long-term engraftment ability of KSL cells. Taken together, these data suggest that TNF-{alpha} produced by FC affect KSL cells in long-term engraftment and survival.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7. FC improve short-term engraftment TNFR–/– KSL cells in allogeneic recipients. A, Life-table survival of B10.BR mice transplanted with allogeneic KSL cells. B10.BR mice were conditioned with 950 cGy total body irradiation, and transplanted with 10,000 B6 KSL cells alone ({square}) or in presence of 30,000 FC from B6 ({blacksquare}) or with 10,000 KSL cells from TNFR–/– mice alone ({triangleup}) or with 30,000 wt FC ({blacktriangleup}). Data represent three different experiments. B, CFC assay preformed with 1,000 freshly sorted KSL cells from B6 or TNFR–/– mice cultured in the presence or the absence of 3,000 B6 FC. Data represent the average of two different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The fact that FC potently enhance engraftment of purified HSC in allogeneic (3) and syngeneic (4) recipients led us to examine whether FC exert a regulatory effect on HSC. We found that FC significantly enhance the clonogenicity and increase the frequency of early and late progenitors (20) generated by KSL cells in vitro using CAFC/LTC-IC assays. FC have previously been reported to increase the frequency of late CAFC on impaired allogeneic stroma (28), further supporting a role for FC in the hemopoietic microenvironment. The effect of FC was not due to an increase in proliferation or differentiation of HSC, because the HSC cocultured with FC retained the ability to generate more multipotent colonies in vitro, as well as to promote long-term engraftment in vivo. Instead, we found that FC act directly on HSC to prevent apoptosis, a function requiring FC:HSC interaction. A clear understanding of how FC enhance the establishment of chimerism and tolerance may have a direct impact on clinical efforts to establish chimerism and treat autoimmune disease states.

The present studies demonstrate that TNF-{alpha} is critical to FC function in vivo and in vitro. TNF-{alpha} is a potent bifunctional regulator of hemopoietic stem and progenitor cells, depending upon additional growth factors present and the maturation stage of the cells (17, 29). TNF-{alpha} inhibits the growth of primitive HSC, preventing entry into S-phase from G0/G1 during stimulation by growth factors (29). Under hemopoietic stress (i.e., after transplantation), low levels of TNF-{alpha} recruit more primitive HSC to proliferate but simultaneously induce a differentiation block downstream to maintain self-renewal (16, 17). We found that FC from TNF-{alpha}–/– mice were significantly impaired in function in vivo as evidenced by loss of facilitative capability for both syngeneic and allogeneic HSC engraftment. The fact that graft failure occurred at an accelerated rate when FC from TNF-{alpha}–/– donors were administered with wt KSL cells would suggest that FC were ineffective in promoting HSC survival without the early production of TNF-{alpha} and/or that interaction of FC with HSC induced activation of specific pathways in HSC that accelerated their demise in the absence of TNF-{alpha}. Studies are underway to elucidate these observations. We confirmed that TNF-{alpha} produced by FC targeted HSC function by using KSL cells from TNFR–/– mice. First, TNFR–/– KSL cells show poor engraftment potential, suggesting that TNF-{alpha} produced by the host microenvironment influences in HSC engraftment. Second, wt FC were only able to help for short-term engraftment, suggesting that FC provide a combination of factors that secure HSC for long-term engraftment, among which TNF-{alpha} plays a major role.

TNF-{alpha}–/– FC were also functionally impaired in vitro, as they did not enhance KSL cell clonogenicity or survival. Blocking TNF-{alpha} on FC before coculture with KSL cells completely abrogated the enhanced clonogenicity and antiapoptotic effects mediated by FC on KSL cells. Although TNF-{alpha} was not detected by cytokine array, neutralization of TNF-{alpha} in the FC significantly impaired the clonogenicity-enhancing effect on HSC. Several studies have demonstrated a dose-dependent effect of TNF-{alpha} on hemopoietic cells in vitro (17, 19, 30, 31), and in most cases, the beneficial regulatory effect occurred at low doses, which corroborates our observations (17). To our knowledge, this is the characterization of a BM cell directly responsible for TNF-{alpha} production after contact with KSL cells which in turn increases survival and maintains multipotentiality of HSC.

Collectively, our findings indicate that reciprocal cross-regulation between KSL cells and FC occurs in the FC effect. The fact that SN from FC cultured with KSL cells replaces FC in promoting KSL cell clonogenicity suggests that KSL cells induce the production of cytokines by FC. Neutralizing TNF-{alpha} in the FC-KSL cell SN negated the effect of enhanced KSL cell clonogenicity, supporting the hypothesis that KSL cells induce FC to produce TNF-{alpha}, and additional cytokines such as IL-6 and IFN-{gamma} may act synergistically with TNF-{alpha} on KSL cells to provide an optimal effect in vivo (32, 33). Furthermore, the fact that FC lose their ability to enhance clonogenicity when separated from HSC by a transwell membrane supports the hypothesis that direct contact between the cells is required for the full biologic effect of FC on HSC. Moreover, although SN from FC cultured with HSC replaced FC in enhancing HSC clonogenicity, it did not replace FC in its antiapoptotic effect on HSC, further supporting a requirement for direct contact between the cells.

HSC death is the major limiting factor for successful engraftment after transplantation (25, 26). Several regulatory factors have been reported to influence HSC survival decisions. Bcl-2, Bcl-xL, and FLIP are cell death repressors found within the HSC at selected stages of differentiation (34, 35, 36, 37). The fact that FC-induced Bcl-3 transcript up-regulation suggests a possible role for the NF-{kappa}B/I{kappa}B pathway in maintaining HSC survival. Bcl-3, a member of the I{kappa}B subfamily of transcriptional regulators, increases the transcriptional activity of NF-{kappa}B family members and is a known transcriptional regulator when associated with NF-{kappa}B family members (38). Bcl-3 also has activities that make it an interesting candidate as a survival factor for HSC. It is often overexpressed in human leukemia and tumors (39), it shows survival activity in lymphocytes without driving cellular proliferation (40), and is strongly induced in growth factor-stimulated erythroid precursors (41). The up-regulation of Bcl-3 transcription in KSL cells cocultured with FC supports an antiapoptotic mechanism of action for FC and is the first indication of a role for Bcl-3 in regulation of HSC survival. Notably, Bcl-2 transcript levels were decreased in KSL cells cocultured with FC. This accessory cell-dependent down-regulation is reminiscent of that seen in activated T cells (42). We found that the production of TNF-{alpha} by FC is required for the up-regulation of Bcl-3 in HSC. Preincubation of FC with anti-TNF-{alpha} before coculture with KSL cells abrogated Bcl-3 transcription in KSL cells. Furthermore, incubation of KSL cells with TNF-{alpha} stimulated Bcl-3 transcription as effectively as FC, confirming a direct role for TNF-{alpha} in this pathway. Although TNF-{alpha} is an apoptotic agent, it can also increase survival (43, 44) by activating NF-{kappa}B (45, 46). NF-{kappa}B is present in all human BM cells and is required for survival as well as for clonogenicity of HSC (46). Although TNF-{alpha} has been shown to induce Bcl-3 up-regulation in B cells as well as in the liver cell line HepG2 while regulating NF-{kappa}B (47), this is the first evidence of such regulation in HSC survival. Our findings suggest a critical role for TNF-{alpha} produced by FC in HSC survival by regulating NF-{kappa}B activity, at least through Bcl-3, because FC increase HSC survival and the frequency of primitive stem cells. These data corroborate previous observations showing that TNF-{alpha} protects quiescent stem cells from apoptosis in vitro (23, 44). The exact role of Bcl-3 in regulation of HSC survival remains to be established. The fact that HSC stimulate FC to produce TNF-{alpha}, which in turn regulates the HSC, further confirms a cross-regulation between HSC and FC.

In conclusion, we show that FC act on the more primitive subpopulations of HSC to promote survival and function via production of physiologically relevant levels of TNF-{alpha}. This finding confirms the regulatory role of accessory cells such as FC in the BM microenvironment. The fact that small numbers of FC exert a significant beneficial effect on HSC in vivo and in vitro, an effect that requires bidirectional cellular cross-talk, may make it a promising cell to establish chimerism and tolerance in the clinic.


   Acknowledgments
 
We thank H. Lee Grimes and Thomas Mitchell for review of the manuscript and helpful comments; Barry Udis, Daniel Cramer, and Lala Hussain for technical assistance; Carolyn DeLautre for manuscript preparation; and the staff of the University of Louisville animal facility for outstanding animal care.


   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 in part by National Institutes of Health (NIH) R01 DK069766 and NIH 5RO1 HL063442; Juvenile Diabetes Research Foundation (JDRF) 1-2005-1037 and JDRF 1-2006-1466; the Department of the Navy, Office of Naval Research N000140610084; the Commonwealth of Kentucky Research Challenge Trust Fund; the W. M. Keck Foundation; The Jewish Hospital Foundation; and the University of Louisville Hospital. Back

2 Address correspondence and reprint requests to Dr. Suzanne T. Ildstad, Institute for Cellular Therapeutics, University of Louisville, 570 South Preston Street, Suite 404, Louisville, KY 40202-1760. E-mail address: stilds01{at}louisville.edu Back

3 Abbreviations used in this paper: BM, bone marrow; BMT, BM transplantation; FC, facilitating cell; HSC, hemopoietic stem cell; p-preDC, precursor plasmacytoid dendritic cell; ODN, oligodeoxynucleotide; LTCM, long-term culture medium; SN, supernatant; CFC, colony-forming cell; CAFC, cobblestone area-forming cell; LTC-IC, long-term culture-initiating cell; 7-AAD, 7-aminoactinomycin D; wt, wild type. Back

Received for publication March 6, 2007. Accepted for publication October 15, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Lu, L., R. N. Shen, H. E. Broxmeyer. 1996. Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: current status and future application. Crit. Rev. Oncol. Hematol. 22: 61-78. [Medline]
  2. Korbling, M., P. Anderlini. 2001. Peripheral blood stem cell versus bone marrow allotransplantation: does the source of hematopoietic stem cells matter?. Blood 98: 2900-2908. [Abstract/Free Full Text]
  3. Kaufman, C. L., Y. L. Colson, S. M. Wren, S. L. Watkins, R. L. Simmons, S. T. Ildstad. 1994. Phenotypic characterization of a novel bone-marrow derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 84: 2436-2446. [Abstract/Free Full Text]
  4. Grimes, H. L., C. L. Schanie, Y. Huang, D. Cramer, F. Rezzoug, I. Fugier-Vivier, S. T. Ildstad. 2004. Graft facilitating cells are derived from hematopoietic stem cells and functionally require CD3, but are distinct from T lymphocytes. Exp. Hematol. 32: 946-954. [Medline]
  5. Fugier-Vivier, I., F. Rezzoug, Y. Huang, A. J. Graul-Layman, C. L. Schanie, H. Xu, P. M. Chilton, S. T. Ildstad. 2005. Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment. J. Exp. Med. 201: 373-383. [Abstract/Free Full Text]
  6. Huang, Y., F. Rezzoug, P. M. Chilton, H. L. Grimes, D. E. Cramer, S. T. Ildstad. 2004. Matching at the MHC class I K locus is essential for long-term engraftment of purified hematopoietic stem cells: a role for host NK cells in regulating HSC engraftment. Blood 104: 873-880. [Abstract/Free Full Text]
  7. Eaves, C. J., J. D. Cashman, R. J. Kay, G. J. Dougherty, T. Otsuka, L. A. Gaboury, D. E. Hogge, P. M. Lansdorp, A. C. Eaves, R. K. Humphries. 1991. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 78: 110-117. [Abstract/Free Full Text]
  8. Calvi, L. M., G. B. Adams, K. W. Weibrecht, J. M. Weber, D. P. Olson, M. C. Knight, R. P. Martin, E. Schipani, P. Divieti, F. R. Bringhurst, et al 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425: 841-846. [Medline]
  9. Lataillade, J. J., D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, M. C. Bousse-Kerdiles. 2002. Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism. Blood 99: 1117-1129. [Abstract/Free Full Text]
  10. Karlsson, R., M. Engstrom, M. Jonsson, P. Karlberg, C. J. Pronk, J. Richter, J. I. Jonsson. 2003. Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes. J. Leukocyte Biol. 74: 923-931. [Abstract/Free Full Text]
  11. Broxmeyer, H. E., S. Cooper, L. Kohli, G. Hangoc, Y. Lee, C. Mantel, D. W. Clapp, C. H. Kim. 2003. Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo. J. Immunol. 170: 421-429. [Abstract/Free Full Text]
  12. Mohle, R., F. Bautz, S. Rafii, M. A. Moore, W. Brugger, L. Kanz. 1998. The chemokine receptor CXCR-4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1. Blood 91: 4523-4530. [Abstract/Free Full Text]
  13. Wright, D. E., E. P. Bowman, A. J. Wagers, E. C. Butcher, I. L. Weissman. 2002. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J. Exp. Med. 195: 1145-1154. [Abstract/Free Full Text]
  14. Matsunaga, T., T. Kato, H. Miyazaki, M. Ogawa. 1998. Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. Blood 92: 452-461. [Abstract/Free Full Text]
  15. Wineman, J. P., S. Nishikawa, C. E. Muller-Sieburg. 1993. Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit. Blood 81: 365-372. [Abstract/Free Full Text]
  16. Caux, C., C. Favre, S. Saeland, V. Duvert, I. Durand, P. Mannoni, J. Banchereau. 1991. Potentiation of early hematopoiesis by tumor necrosis factor-{alpha} is followed by inhibition of granulopoietic differentiation and proliferation. Blood 78: 635-644. [Abstract/Free Full Text]
  17. Snoeck, H. W., S. Weekx, A. Moulijn, F. Lardon, M. Lenjou, G. Nys, P. C. Van Ranst, D. R. Van Bockstaele, Z. N. Berneman. 1996. Tumor necrosis factor {alpha} is a potent synergistic factor for the proliferation of primitive human hematopoietic progenitor cells and induces resistance to transforming growth factor β but not to interferon {gamma}. J. Exp. Med. 183: 705-710. [Abstract/Free Full Text]
  18. Jacobsen, S. E., F. W. Ruscetti, C. M. Dubois, J. R. Keller. 1992. Tumor necrosis factor {alpha} directly and indirectly regulates hematopoietic progenitor cell proliferation: role of colony-stimulating factor receptor modulation. J. Exp. Med. 175: 1759-1772. [Abstract/Free Full Text]
  19. Rusten, L. S., F. W. Jacobsen, W. Lesslauer, H. Loetscher, E. B. Smeland, S. E. Jacobsen. 1994. Bifunctional effects of tumor necrosis factor {alpha} (TNF {alpha}) on the growth of mature and primitive human hematopoietic progenitor cells: involvement of p55 and p75 TNF receptors. Blood 83: 3152-3159. [Abstract/Free Full Text]
  20. Ploemacher, R. E., J. P. van der Sluijs, J. A. Voerman, N. C. Brons. 1989. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 74: 2755-2763. [Abstract/Free Full Text]
  21. Cheng, T., N. Rodrigues, H. Shen, Y. Yang, D. Dombkowski, M. Sykes, D. T. Scadden. 2000. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287: 1804-1808. [Abstract/Free Full Text]
  22. de Haan, G., S. J. Szilvassy, T. E. Meyerrose, B. Dontje, B. Grimes, G. Van Zant. 2000. Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers. Blood 96: 1374-1379. [Abstract/Free Full Text]
  23. Ratajczak, J., M. Kucia, R. Reca, J. Zhang, B. Machalinski, M. Z. Ratajczak. 2003. Quiescent CD34+ early erythroid progenitors are resistant to several erythropoietic "inhibitory" cytokines: role of FLIP. Br. J. Haematol. 123: 160-169. [Medline]
  24. Domen, J., S. H. Cheshier, I. L. Weissman. 2000. The role of apoptosis in the regulation of hematopoietic stem cells: overexpression of Bcl-2 increases both their number and repopulation potential. J. Exp. Med. 191: 253-264. [Abstract/Free Full Text]
  25. Liu, B., S. M. Buckley, I. D. Lewis, A. I. Goldman, J. E. Wagner, J. C. van der Loo. 2003. Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95. Exp. Hematol. 31: 824-832. [Medline]
  26. Mata, M., E. Chiffoleau, S. H. Adler, T. Gray, W. Hancock, L. A. Turka. 2004. Bcl-xL expression in stem cells facilitates engraftment and reduces the need for host conditioning during bone marrow transplantation. Am. J. Transplant. 4: 58-64. [Medline]
  27. Brawand, P., D. R. Fitzpatrick, B. W. Greenfield, K. Brasel, C. R. Maliszewski, T. De Smedt. 2002. Murine plasmacytoid pre-dendritic cells generated from Flt3 ligand-supplemented bone marrow cultures are immature APCs. J. Immunol. 169: 6711-6719. [Abstract/Free Full Text]
  28. Yaroslavskiy, B., Y. L. Colson, S. T. Ildstad, D. Parrish, S. S. Boggs. 1998. Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma. Exp. Hematol. 26: 604-611. [Medline]
  29. Zhang, Y., A. Harada, H. Bluethmann, J. B. Wang, S. Nakao, N. Mukaida, K. Matsushima. 1995. Tumor necrosis factor (TNF) is a physiologic regulator of hematopoietic progenitor cells: increase of early hematopoietic progenitor cells in TNF receptor p55-deficient mice in vivo and potent inhibition of progenitor cell proliferation by TNF {alpha} in vitro. Blood 86: 2930-2937. [Abstract/Free Full Text]
  30. Broxmeyer, H. E., D. E. Williams, L. Lu, S. Cooper, S. L. Anderson, G. S. Beyer, R. Hoffman, B. Y. Rubin. 1986. The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-{gamma}. J. Immunol. 136: 4487-4495. [Abstract]
  31. Maguer-Satta, V., R. Oostendorp, D. Reid, C. J. Eaves. 2000. Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates. Blood 96: 4118-4123. [Abstract/Free Full Text]
  32. Szilvassy, S. J., T. E. Meyerrose, B. Grimes. 2000. Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells. Blood 95: 2829-2837. [Abstract/Free Full Text]
  33. Gothot, A., J. C. van der Loo, D. W. Clapp, E. F. Srour. 1998. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice. Blood 92: 2641-2649. [Abstract/Free Full Text]
  34. Peters, R., S. Leyvraz, L. Perey. 1998. Apoptotic regulation in primitive hematopoietic precursors. Blood 92: 2041-2052. [Abstract/Free Full Text]
  35. Ogilvy, S., D. Metcalf, C. G. Print, M. L. Bath, A. W. Harris, J. M. Adams. 1999. Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival. Proc. Natl. Acad. Sci. USA 96: 14943-14948. [Abstract/Free Full Text]
  36. Park, J. R., I. D. Bernstein, D. M. Hockenbery. 1995. Primitive human hematopoietic precursors express Bcl-x but not Bcl-2. Blood 86: 868-876. [Abstract/Free Full Text]
  37. Kim, H., K. A. Whartenby, R. W. Georgantas, III, J. Wingard, C. I. Civin. 2002. Human CD34+ hematopoietic stem/progenitor cells express high levels of FLIP and are resistant to Fas-mediated apoptosis. Stem Cells 20: 174-182. [Abstract/Free Full Text]
  38. Bours, V., G. Franzoso, V. Azarenko, S. Park, T. Kanno, K. Brown, U. Siebenlist. 1993. The oncoprotein Bcl-3 directly transactivates through {kappa}B motifs via association with DNA-binding p50B homodimers. Cell 72: 729-739. [Medline]
  39. Cogswell, P. C., D. C. Guttridge, W. K. Funkhouser, A. S. Baldwin, Jr. 2000. Selective activation of NF-{kappa}B subunits in human breast cancer: potential roles for NF-{kappa}B2/p52 and for Bcl-3. Oncogene 19: 1123-1131. [Medline]
  40. Mitchell, T. C., D. Hildeman, R. M. Kedl, T. K. Teague, B. C. Schaefer, J. White, Y. Zhu, J. Kappler, P. Marrack. 2001. Immunological adjuvants promote activated T cell survival via induction of Bcl-3. Nat. Immunol. 2: 397-402. [Medline]
  41. Zhang, M. Y., E. W. Harhaj, L. Bell, S. C. Sun, B. A. Miller. 1998. Bcl-3 expression and nuclear translocation are induced by granulocyte-macrophage colony-stimulating factor and erythropoietin in proliferating human erythroid precursors. Blood 92: 1225-1234. [Abstract/Free Full Text]
  42. Mitchell, T. C., T. K. Teague, D. A. Hildeman, J. Bender, W. A. Rees, R. M. Kedl, B. Swanson, J. W. Kappler, P. Marrack. 2002. Stronger correlation of bcl-3 than bcl-2, bcl-xL, costimulation, or antioxidants with adjuvant-induced T cell survival. Ann. NY Acad. Sci. 975: 114-131. [Medline]
  43. Dempsey, P. W., S. E. Doyle, J. Q. He, G. Cheng. 2003. The signaling adaptors and pathways activated by TNF superfamily. Cytokine Growth Factor Rev. 14: 193-209. [Medline]
  44. Bryder, D., V. Ramsfjell, I. Dybedal, K. Theilgaard-Monch, C. M. Hogerkorp, J. Adolfsson, O. J. Borge, S. E. Jacobsen. 2001. Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J. Exp. Med. 194: 941-952. [Abstract/Free Full Text]
  45. Kerzic, P. J., D. W. Pyatt, J. H. Zheng, S. A. Gross, A. Le, R. D. Irons. 2003. Inhibition of NF-{kappa}B by hydroquinone sensitizes human bone marrow progenitor cells to TNF-{alpha}-induced apoptosis. Toxicology 187: 127-137. [Medline]
  46. Pyatt, D. W., W. S. Stillman, Y. Yang, S. Gross, J. H. Zheng, R. D. Irons. 1999. An essential role for NF-{kappa}B in human CD34+ bone marrow cell survival. Blood 93: 3302-3308. [Abstract/Free Full Text]
  47. Brasier, A. R., M. Lu, T. Hai, Y. Lu, I. Boldogh. 2001. NF-{kappa}B-inducible BCL-3 expression is an autoregulatory loop controlling nuclear p50/NF-{kappa}B1 residence. J. Biol. Chem. 276: 32080-32093. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 DiabetesHome page
Y. Huang, I. J. Fugier-Vivier, T. Miller, M. J. Elliott, H. Xu, L. D. Bozulic, P. M. Chilton, and S. T. Ildstad
Plasmacytoid Precursor Dendritic Cells From NOD Mice Exhibit Impaired Function: Are They a Component of Diabetes Pathogenesis?
Diabetes, September 1, 2008; 57(9): 2360 - 2370.
[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 Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rezzoug, F.
Right arrow Articles by Ildstad, S. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rezzoug, F.
Right arrow Articles by Ildstad, S. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS