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International Immunology Advance Access originally published online on March 29, 2006
International Immunology 2006 18(5):653-659; doi:10.1093/intimm/dxl002
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

NF-{kappa}B is dispensable for normal lymphocyte development in bone marrow but required for protection of progenitors from TNF{alpha}

Hideya Igarashi1,*, Yoshihiro Baba1,*, Yoshinori Nagai1, Eijiro Jimi2, Sankar Ghosh2 and Paul W Kincade1

1 Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
2 Section of Immunobiology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA

Correspondence to: P. W. Kincade; E-mail: kincade{at}omrf.ouhsc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and Discussion
 References
 
Levels of the nuclear factor-kappa B (NF-{kappa}B)/Rel family of proteins are carefully modulated in differentiating lymphocytes, where these transcription factors are thought to be important for survival and fate decisions. In contrast, gene-targeting experiments have not revealed clear roles for these transcription factors in lymphopoiesis within bone marrow. Inhibition of NF-{kappa}B by introduction of mutated I{kappa}B{alpha}, a ‘superinhibitor’ of NF-{kappa}B, into hematopoietic stem cells or early progenitors suppressed B as well as T lymphopoiesis following transplantation into immunodeficient mice. Furthermore, a NF-{kappa}B essential modifier-binding domain (NBD) peptide that blocks IKB kinase (IKK) activity selectively impaired the generation of adult B lineage cells. However, this suppression did not occur when a neutralizing antibody to tumor necrosis factor {alpha} (TNF{alpha}) was added to the cultures, or in circumstances where few non-lymphoid cells were present. We conclude that while NF-{kappa}B plays a survival-promoting role in lymphoid progenitors, this may only be significant in circumstances such as transplantation when levels of TNF{alpha} are high.

Keywords: adult bone marrow, fetal liver, lymphopoiesis, NF-{kappa}B, TNF{alpha}


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and Discussion
 References
 
Although nuclear factor-kappa B (NF-{kappa}B) was initially discovered in a pre-B lymphoma cell line, remarkably little has been known about its role in B lymphopoiesis. Mice lacking various NF-{kappa}B/Rel family members, including p50, p52, c-Rel or RelB have displayed no marked impairment of lymphocyte development (1). This appeared to be the case for p50/p65 double-knockout mice where E12 fetal liver cells were used as a source for hematopoietic progenitor cells because of embryonic lethality (2). When mixed equal parts with wild-type fetal liver cells, p50–/–/p65–/– cells had normal differentiation potential. Similarly, irradiation chimeras of IKKß–/– fetal liver cells had a complete block in B and T cell generation (3). Again, these hematopoietic precursors developed into mature B and T cells when wild-type bone marrow cells were simultaneously transferred. While NF-{kappa}B essential modifier (NEMO)-deficient fetal liver cells failed to reconstitute B and T lineage cells in irradiated recipients, NEMO–/– embryonic stem cells differentiated into B lineage cells when placed on OP9 stromal cells (4). Prendes et al. (5) demonstrated that B cell generation from RelA (p65)–/– fetal liver was greatly diminished in transplanted mice while lymphopoietic cells from RelA–/–TNFR{alpha}–/– double-knockout mice were normal. This study strongly suggests that NF-{kappa}B plays a role in B cell survival by inhibiting tumor necrosis factor {alpha} (TNF{alpha}) cytotoxity.

Therefore, it is unlikely that fetal lymphoid cells were intrinsically defective in these gene-targeted animals. However, lymphopoiesis in normal adults is substantially different from that occurring in fetal life (6) and lymphoid progenitors from NF-{kappa}B-deficient adults have not been well studied. Two recent studies used retroviral transduction or transgenic mice with an I{kappa}B{alpha} mutated inhibitor to conclude that NF-{kappa}B may indeed be important for B lymphopoiesis (7, 8). We have now exploited a series of approaches to investigate the importance of NF-{kappa}B to fetal and adult lymphopoiesis. Retroviral introduction of constitutively active I{kappa}B{alpha} into lympho-hematopoietic progenitors before transplantation to irradiated immunodeficient mice indicated that the NF-{kappa}B/Rel pathway might be crucial for B as well as T lymphopoiesis. Furthermore, differentiation of adult lymphoid progenitors was blocked by addition of a NEMO-binding domain (NBD) peptide to cultures. However, additional experiments indicate that while NF-{kappa}B-deficient progenitors are particularly sensitive to TNF{alpha}, they retain normal potential for lymphocyte generation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and Discussion
 References
 
Mice
The congenic strains of mice, C57BL/6J and recombination-activating gene (RAG) 1-deficient mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in our specific pathogen-free laboratory animal facility. NF-{kappa}B/luciferase reporter gene transgenic B10A mice bearing two NF-{kappa}B-binding sites were described elsewhere (8). We backcrossed these mice more than three times to a C57BL/6J background before use in these studies.

Luciferase assay
NF-{kappa}B activity was measured in cells from luciferase reporter gene transgenic mice. The cells were harvested and lysed with passive buffer (Promega, Madison, WI, USA) at 1 x 105 cells 25 µl–1 on ice for 15 min, then centrifuged for 5 min at reverse transcription to sediment cell debris. The cell lysate was then mixed with 100 µl of luciferase substrate (Promega) and luciferase activity was measured with a luminometer for 30 s per sample.

Lineage cell depletion and cell sorting
Bone marrow or E15 fetal liver cells were enriched for lineage negative-cells by incubation with antibodies to lineage markers, anti-Gr-1 (Ly-6G, RB6-8C5) and anti-CD11b/Mac-1 (M1/70) for myeloid cells, anti-CD19 (1D3) and anti-CD45R/B220 (RA3/6B2) for B lineage cells and anti-Ter-119 for erythroid cells, followed by negative selection using the MACS cell separation system (Miltenyi Biotech, Auburn, CA, USA). The same antibodies without CD11b/Mac-1 were used for lineage depletion of fetal liver. These partially lineage-depleted cells were further stained with FITC–anti-lineage markers [Gr-1, Mac-1 (adult only), CD2; LFA-2, CD3; 145-2C11, CD8a; 53-6.7, Ter-119 and CD45R] as well as PE–anti-Sca-1antibody (Ly6A/E, E13-161.7) and allophycoerythrin (APC)–anti-c-kit (2B8). They were then sorted on a MoFlo (DakoCytomation, Fort Collins, CO, USA). APC–anti-CD11b/Mac-1, PE–anti-CD19, PE–anti-pan-NK marker (DX5), PE–anti-CD4 (L3T4) and APC–anti-TCRß (H57-597) were used for flow cytometry analysis of cells recovered from cultures. All mAbs were purchased from BD PharMingen. Flow cytometry was performed on a FACSCalibur (Becton Dickinson, Mountain View, CA, USA), and the data were analyzed with Flowjo software (Treestar, San Carlos, CA, USA).

Retrovirus-mediated gene transfer
The cDNA for a previously described NF-{kappa}B superinhibitor (SI) cassette was cloned into the MigR1 retrovirus vector kindly provided by Xiao-Hong Sun (Oklahoma Medical Research Foundation) using an EcoRI site. This vector was designed to express protein from inserted cDNA and green fluorescent protein (GFP) independently (9). Three micrograms of retrovirus construct were introduced to 1 x 106 cells of a packaging cell line (EcoPack2: BD Biosciences Clontech, Palo Alto, CA, USA) in 60-mm culture dishes using a liposome method (FuGene 6: Roche Molecular Biochemicals, Indianapolis, IN, USA). The culture medium (3 ml) was changed once 24 h post-transfection and then virus-containing supernatant was collected 48 h later. Purified Lin– c-kitLo Sca-1Lo or Lin– c-kitHi Sca-1Hi cells were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 15% FCS and 1% detoxified BSA (Stem Cell Technologies, Vancouver, British Columbia, Canada) for 48 h prior to infection in the presence of recombinant mouse stem cell factor (SCF) (100 ng ml–1), Flk-2/Flt3L (100 ng ml–1), IL-7 (1 ng ml–1) and IL-11 (10 ng ml–1) as growth factors. One million target cells were pipetted into 5-ml polystyrene tubes. After collecting cells by centrifugation, medium was replaced with 0.5 ml of virus supernatant and an equal volume of fresh medium containing growth factors and 6 µg ml–1 of polybrene (Sigma, St Louis, MO, USA) in a total 1 ml volume. Spin infection was conducted for 45 min at 1800 r.p.m. in a centrifuge at room temperature. The virus cocktail was aspirated and replaced with complete DMEM and growth factors. Cells were re-suspended and transferred to 24-well tissue culture plates and incubated at 37°C for 2 h. Culture medium was aspirated and replaced with a fresh media/virus/polybrene cocktail. Cells were centrifuged for 1 h at 2000 r.p.m. in a centrifuge at room temperature. The virus cocktail was replaced with fresh media and incubated at 37°C for 2 h. Again, culture medium was replaced with fresh media/virus/polybrene cocktail and cultured for 18 h. After incubation, cells were collected in 5-ml tubes and washed twice with PBS to remove polybrene. Cells were cultured in six-well plates with media containing growth factors for an additional 72 h. After culture, cells were sorted into GFP+ populations and used for bone marrow transplantation.

Adoptive transfer
Five thousand purified GFP+ cells were injected intravenously into sub-lethally (500 rad) or lethally (1000 rad) irradiated RAG1-deficient mice. Four weeks later, bone marrow cells, splenocytes and thymocytes were harvested from these mice and the chimerism was analyzed by flow cytometry.

Cell cultures and peptide inhibition
Details for stromal cell-free, serum-free culture of early lymphocyte progenitors are described elsewhere (10). Briefly, sorted cells were cultured with X-VIVO15 medium (Bio Whittaker, Walkersville, MA, USA) containing 1% detoxified BSA (Stem Cell Technologies), 2 mM L-glutamine, 5 x 10–5 M 2-mercaptoethanol, 100 U ml–1 penicillin and 100 mg ml–1 streptomycin. Recombinant mouse SCF (20 ng ml–1), Flk-2/Flt3 ligand (100 ng ml–1) and IL-7 (1 ng ml–1) were included to drive B lymphoid lineage differentiation. To examine TNF{alpha} sensitivity, recombinant mouse TNF{alpha} was added at the indicated concentrations. All cytokines were purchased from R&D Systems (Minneapolis, MN, USA). The NF-{kappa}B inhibitory NBD and mutated control peptides have been described elsewhere (11). They were added to a final concentration of 25 µM. In some experiments, a neutralizing antibody to TNF{alpha} (eBioscience, San Diego, CA, USA) was added to a final concentration of 10 µg ml–1.


   Results and Discussion
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
References
 
Retroviral introduction of an NF-{kappa}B inhibitor blocks lymphopoiesis from adult bone marrow and fetal liver cells
Previous studies have demonstrated that NF-{kappa}B activation can be selectively blocked in T lymphoma cells by expression of a constitutively active and artificially stable human I{kappa}B{alpha} protein (12). To prevent signal-induced degradation, serines 32 and 36 of I{kappa}B{alpha}, which are normally phosphorylated by the I{kappa}B kinases IKK{alpha} and IKKß, were mutated into alanines. In addition, the lysine 21 and 22 sites of ubiquitination were changed to arginines. Tyrosine 42 was mutated to phenylalanine because phosphorylation at this residue has been reported to cause dissociation of NF-{kappa}B from I{kappa}B{alpha} (13). Finally, serines and threonines within the C-terminal PEST region of I{kappa}B{alpha} were mutated into alanines to lower the rate of basal protein turnover (12).

We then utilized a retroviral vector system to introduce this SI into lympho-hematopoietic cells (Fig. 1A). The mutated I{kappa}B{alpha} cDNA along with a haemagglutinin tag at its N-terminal position were inserted into the cloning site of MigR1 retrovirus vector. This allows the simultaneous but independent expression of protein encoded by inserted cDNA and GFP. A control MigR1 retroviral construct encoded GFP alone. Three days after retroviral infection, ~50% of the cells became green (Fig. 1B). To evaluate the reconstitution potential of these cells for lymphoid lineage differentiation, GFP+ cells were sorted and transplanted into sub-lethally irradiated immunodeficient RAG1–/– mice. Significant numbers of donor type B, T and NK lineage cells were generated in mice transplanted with MigR1-transduced GFP+ cells (Fig. 2A). In contrast, no GFP+ lymphocytes were produced from GFP+ progenitors successfully transduced with the inhibitory Mig I{kappa}B{alpha} SI construct. Thymocytes were examined from the same animals and this revealed a nearly complete block at the CD4/CD8 double negative stage (Fig. 2B).


Figure 1
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  		Fig. 1. Introduction of a NF-{kappa}B SI to lymphoid progenitors by retrovirus infection. (A) The infection protocol scheme is illustrated. A NF-{kappa}B SI was inserted in a MigR1 retrovirus vector which produces GFP and proteins encoded by introduced cDNA. The lymphoid progenitors expressing NF-{kappa}B SI were identified as GFP-positive cells and isolated by cell sorting. Sorted GFP-positive cells were then transferred intravenously to irradiated RAG1 knockout mice. (B) Approximately 50% of the cultured cells were GFP+ 3 days post-infection with either the control MigR1 or Mig I{kappa}B{alpha} SI retroviral vectors.

 

Figure 2
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  		Fig. 2. Inhibition of lymphocyte generation of adult bone marrow cells transduced with NF-{kappa}B SI in reconstituted mice. (A) Five thousand GFP+ cells were sorted from MigR1 or Mig I{kappa}B{alpha} SI introduced adult bone marrow Lin– c-kitLo Sca-1Lo lymphoid progenitor cultures and intravenously transplanted to sub-lethally irradiated RAG1–/– mice. Four weeks after transfer, splenocytes were harvested from recipient mice and stained with CD45R for B lineage, TCRß for T lineage and DX5 for NK lineage cells. The stained cells were resolved according to lineage markers versus GFP by flow cytometry. (B) Thymuses from recipient mice were resolved with CD4 and CD8 by flow cytometry. (C) The hematopoietic stem cell containing Lin– c-kitHi Sca-1Hi population of bone marrow was used for reterovirus target cells. Four weeks after transfer to lethally irradiated RAG1–/– mice, bone marrow cells were harvested and analyzed for B and myeloid lineage cell generation by cell-surface staining with CD19 and CD11b/Mac-1.

 
Myelopoiesis from donor stem cells was inefficient in this transplantation model, so additional experiments utilized lethally irradiated RAG1–/– recipients and evaluation of the bone marrow (Fig. 2C). CD11b+ myeloid cells were generated normally from transplanted cells regardless of whether they were transduced with the control MigR1 or inhibitory Mig I{kappa}B{alpha} SI constructs as indicated by the appearance of GFP+ cells in the spleen. Again, production of CD19+ in the bone marrow was totally dependent on NF-{kappa}B activity. These findings contrast with a previous report that NF-{kappa}B deficiency did not affect T cell development (7). This might be because a less efficient I{kappa}B{alpha} mutant (deletion of only the N-terminal region) was used to inhibit NF-{kappa}B activity or that the target cells (5-FU-treated whole bone marrow) were slightly different.

We then used the same approach to explore the importance of NF-{kappa}B for fetal lymphopoiesis. A Lin– c-kitHi Sca-1+ HSC-enriched fraction of E15 fetal liver was sorted and transduced with control MigR1 or inhibitory Mig I{kappa}B{alpha} SI before transplantation of GFP+ cells into lethally irradiated RAG1–/– mice. When examined 4 weeks post-transplantation, Mig I{kappa}B{alpha} SI construct bearing cells yielded reduced percentages, and absolute numbers, of B, T and NK cells in spleens when compared with control MigR1 cells (Fig. 3A). Lymphoid cells were also suppressed in the thymus and bone marrow (Fig 3B and C). While reductions in total numbers of B lymphocytes in spleen ranged from 100- to 140-fold, CD11b/Mac-1+ myeloid cells were 4- to 9-fold less than those emerging from control vector-treated transplants.


Figure 3
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  		Fig. 3. Inhibition of lymphocyte generation from fetal liver cells transduced with NF-{kappa}B SI in reconstituted mice. (A) Five thousand GFP+ cells were sorted from MigR1 or Mig I{kappa}B{alpha} SI introduced Lin– c-kitHi Sca-1Hi populations from E15 fetal liver and intravenously transplanted to lethally irradiated RAG1–/– mice. Splenocytes were harvested from recipient mice and stained with CD45R for B lineage, TCRß for T lineage and DX5 for NK lineage cells. The stained cells were resolved according to lineage markers versus GFP by flow cytometry. (B) Thymuses from recipient mice were resolved by flow cytometry after staining with CD4 and CD8. (C) Bone marrow cells were harvested and analyzed for B and myeloid lineage cell generation by cell-surface staining with CD19 and CD11b/Mac-1.

 
While these results support several previous reports (2, 3, 7) in suggesting that the NF-{kappa}B/Rel family of transcription factors is essential for B, T and NK lineage differentiation, other experimental approaches used previously only revealed a significant requirement for NF-{kappa}B at later stages of development. In some studies, the effect of NF-{kappa}B was limited to the transition from immature T1/T2 cells to mature follicular B cells (13–18). Elevation of NF-{kappa}B appears to occur at that stage through action of the B-lymphocyte stimulator (Blys)/B cell-activating factor of the TNF family (BAFF) cytokine and a NEMO-independent pathway of NF-{kappa}B activation (19–21). Another recent paper demonstrated up-regulation of NF-{kappa}B in large pre-B cells (8) and our own results would be consistent with an earlier requirement for low levels of NF-{kappa}B.

An NF-{kappa}B inhibitory peptide suppresses lymphocyte differentiation
As an independent assessment of the importance of NF-{kappa}B activity for lymphoid differentiation, we used an inhibitory peptide designated NBD in culture experiments (11). This peptide competitively blocks the binding of I{kappa}B kinase IKKß with IKK{gamma}/NEMO. IKK complex formation is crucial for IKK activation, subsequent I{kappa}B degradation and NF-{kappa}B activation (22). We isolated Lin– c-kitLo Sca-1Lo bone marrow cells from transgenic NF-{kappa}B activity reporter mice by sorting and then cultured them for 7 days with or without this peptide. In control cultures where only the dimethyl sulfoxide vehicle was added, luciferase levels reflecting the transcriptional activity of NF-{kappa}B binding to recognition sites in the reporter construct increased 35% within 7 days (Fig. 4A). In contrast, this induction of NF-{kappa}B was almost completely blocked in peptide-treated cultures.


Figure 4
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  		Fig. 4. A peptide inhibitor of NF-{kappa}B selectively suppresses in vitro B cell generation from lymphoid progenitors of adult bone marrow but not fetal liver. (A) Lymphoid progenitors (Lin– c-kitLo Sca-1Lo) from NF-{kappa}B indicator transgenic (open bars) or wild-type mice (black bars) were purified by sorting from adult bone marrow and cultured in serum- and stromal cell-free media containing recombinant mouse SCF, Flk-2/Flt3L and IL-7 in the presence of 25 µM NBD or control (Mut. NBD) peptide. After 7 days, luciferase activity was measured. (B) Lin– c-kitLo Sca-1Lo cells were purified by sorting from adult bone marrow and cultured under the same conditions described in (A). B lineage cell differentiation was assessed by detection of CD19+ expression by flow cytometry after 7 days in culture. (C) Sorted lymphoid progenitors from adult bone marrow or E15.0 fetal liver were cultured under the same conditions described in (A). Yields of total and CD19+ lymphoid cells were calculated by simply dividing numbers of total cells and CD19+ cells recovered by the numbers of progenitors used to initiate the cultures.

 
The inhibitory NBD peptide also selectively suppressed generation of CD19+ lymphocytes from Lin– c-kitLo Sca-1Lo progenitors in stromal cell-free, serum-free culture (Fig. 4B). Production of CD11b+ myeloid cells was unaffected by NBD and a mutated peptide lacking binding ability to IKKß because amino acid replacement had no influence. The selectivity of suppression was even more obvious when absolute numbers of cells recovered from cultures were calculated (Fig. 4C). Parallel cultures were initiated with Lin– c-kitLo Sca-1Lo progenitors from fetal liver (Fig. 4C). Although there was a slight reduction in numbers of total cells recovered from those cultures, the peptide did not specifically suppress lymphopoiesis from fetal cells.

These observations supported the results presented above in indicating that early stages of B and NK lineage differentiation in adult bone marrow are NF-{kappa}B dependent. Effective inhibition with the NBD peptide also suggested that the canonical NEMO-dependent activation pathway is utilized. However, fetal progenitors were curiously resistant to the inhibitory peptide and lymphopoiesis was not suppressed in other cultures when few non-lymphoid cells were present (see below).

B lymphopoiesis in culture is NF-{kappa}B independent in the absence of TNF{alpha}
Several pieces of evidence suggested that TNF{alpha} may be involved in these experimental models and could explain why cultures of fetal liver were not suppressed by the inhibitory peptide. Firstly, the subnormal B lymphopoesis in RelA (p65)–/– fetal liver-transplanted mice was not seen when RelA–/–TNFR{alpha}–/– fetal liver cells were used (5). We found in other studies that Mac-1+ cells could produce TNF{alpha} in bone marrow cultures (23) and adult bone marrow cells tended to generate more Mac-1+ cells than did fetal liver cells. Therefore, adult bone marrow cells were placed in culture in the presence or absence of a TNF{alpha} neutralizing antibody (Fig. 5A). As before, we observed very specific, peptide-mediated suppression of B lymphopoiesis in control cultures. However, this response was completely blocked by neutralization of TNF{alpha}. The participation of TNF{alpha} in this response was even more obvious when the data were calculated in terms of absolute yields of lymphoid or myeloid cells (Fig. 5B).


Figure 5
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  		Fig. 5. Dependence of lymphoid progenitors on NF-{kappa}B is only apparent in the presence of TNF{alpha}. (A) Lin– c-kitHi Sca-1+ cells from adult bone marrow were sorted and cultured in serum- and stromal cell-free media containing recombinant mouse SCF, Flk-2/Flt3L and IL-7. As indicated, 25 µM NBD or control peptides and/or 10 µg ml–1 anti-TNF{alpha} antibody was added. After 12 days, CD19+ lymphocytes and CD11b/Mac-1+ myeloid cells were assessed by flow cytometry. (B) Yields of each cell type per input progenitor are shown. Similar results were obtained in six independent experiments. Significant differences are indicated by P < 0.001. Statistical significance was determined through Student's t-testing. (C) Sorted Lin– c-kitHi Sca-1+ cells from adult bone marrow or E15.0 fetal liver were cultured under the same conditions described in (A) in the presence of recombinant TNF{alpha}. B and myeloid lineage cell differentiation was assessed by detection of CD19 and CD11b/Mac-1 after 15 days in culture. (D) Yields of each cell type per input progenitor are shown. Similar results were obtained in two independent experiments. Error bars represent standard deviations of the mean. Significant differences are indicated by an asterisk (P < 0.002) or two asterisks (P < 0.02). Statistical significance was determined by Student's t-test.

 
Additional experiments were performed to test the cytokine sensitivity of fetal and adult lymphoid progenitors. Both were suppressed by TNF{alpha} in a dosage-dependent manner (Fig. 5C and D). Lin– c-kitLo Flk-2+ IL-7R{alpha}+ progenitors from BALB/C marrow or Lin– c-kitLo Sca-1+ IL-7R{alpha}+ cells from C57BL/6 marrow were then isolated and placed in culture. Almost no CD11b/Mac-1+ cells were produced under these circumstances, and there was no inhibition of lymphopoiesis by addition of the NBD peptide (data not shown). Furthermore, the Lin– c-kitHi Sca-1Hi HSC-rich fraction of adult bone marrow generated B lineage lymphocytes normally when transduced with the NF-{kappa}B SI and placed on OP9 stromal cells for 2 weeks (data not shown).

Collectively, these results indicate that NF-{kappa}B is important for lymphocyte development, but only in circumstances such as inflammation or transplantation where TNF{alpha} is present. Depletion of TNF{alpha} from culture of normal progenitors had no impact on the efficiency of B lineage cell generation (Fig. 5A). This is consistent with the fact that no lymphocyte developmental abnormalities were found in TNFR, TNF{alpha}, and RelA/TNFR double-deficient mice (24, 25). Myeloid cells are the most likely source of TNF{alpha} in myeloid cell containing cultures and the cytokine is probably elevated in irradiated mice. TNF{alpha} can elicit caspase activation and cell death in lymphoid progenitors if not adequately compensated by NF-{kappa}B activation.

Steroid hormones inhibit lymphopoiesis from the earliest stages and NF-{kappa}B-dependent mechanisms for hormone action have been described (3, 26, 27). However, our new findings indicate that depletion of NF-{kappa}B would alone be insufficient to suppress lymphopoiesis. The observations further suggest that it would be desirable to maintain NF-{kappa}B levels during transplantation, regeneration from chemotherapy or any other circumstance where TNF{alpha} might be present. Otherwise, recovery of the immune system could be compromised.


   Acknowledgements
 
We acknowledge expert technical assistance provided by Karla P. Garrett, Sophia C. Gregory, Jacob Bass, Diana Hamilton and Viji Dandapani. Finally, we appreciate the secretarial help provided by Shelli Wasson. This work was supported by grants AI20069, AI58162, P20RR15577 and AI33443 (S.G.) from the National Institutes of Health. P.W.K. holds the William H. and Rita Bell Chair in biomedical research.


   Abbreviations
 
APC, allophycoerythrin
DMEM, Dulbecco's modified eagle medium
GFP, green fluorescent protein
IKK, IKB kinase
NBD, NEMO-binding domain
NEMO, NF-{kappa}B essential modifier
NF-{kappa}B, nuclear factor-kappa B
RAG, recombination-activating gene
SCF, stem cell factor
TNF{alpha}, tumor necrosis factor {alpha}

   Notes
 
* These authors contributed equally to this work. Back

Transmitting editor: T. F. Tedder

Received 1 July 2005, accepted 27 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results and Discussion
 References
 

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F. Guo, S. Tanzer, M. Busslinger, and F. Weih
Lack of nuclear factor-{kappa}B2/p100 causes a RelB-dependent block in early B lymphopoiesis
Blood, August 1, 2008; 112(3): 551 - 559.
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