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. Author manuscript; available in PMC: 2012 Sep 10.
Published in final edited form as: Exp Hematol. 2008 May;36(5):545–558. doi: 10.1016/j.exphem.2007.12.012

TNFα and Endothelial Cells Modulate Notch Signaling in the Bone Marrow Microenvironment during Inflammation

Luis Fernandez a, Sonia Rodriguez b,*, Hui Huang a,*, Angelo Chora c, Christin Mumaw b, Eugenia Cruz d, Karen Pollok b, Filipa Cristina c, Joanne E Price e, Michael J Ferkowicz b, David T Scadden a, Matthias Clauss f, Angelo A Cardoso g, Nadia Carlesso a,b,**
PMCID: PMC3437760  NIHMSID: NIHMS141648  PMID: 18439488

Abstract

Objective

Homeostasis of the hematopoietic compartment is challenged and maintained during conditions of stress by mechanisms that are poorly defined. To understand how the bone marrow (BM) microenvironment influences hematopoiesis, we explored the role of Notch signaling and bone marrow endothelial cells in providing microenvironmental cues to hematopoietic cells in the presence of inflammatory stimuli.

Methods

The human BM endothelial cell line BMEC and primary human BM endothelial cells were analyzed for expression of Notch ligands and the ability to expand hematopoietic progenitors in an in vitro co-culture system. In vivo experiments were carried out to identify modulation of Notch signaling in BM endothelial and hematopoietic cells in mice challenged with TNFα or LPS, or in Tie2-tmTNFα transgenic mice characterized by constitutive TNFα activation.

Results

BM endothelial cells were found to express Jagged ligands and to greatly support progenitor’s colony-forming ability. This effect was markedly decreased by Notch antagonists and augmented by increasing levels of Jagged2. Physiologic upregulation of Jagged2 expression on BMEC was observed upon TNFα activation. Injection of TNFα or LPS upregulated 3 to 4 fold Jagged2 expression on murine BM endothelial cells in vivo and resulted in increased Notch activation on murine hematopoietic stem/progenitor cells. Similarly, constitutive activation of endothelial cells in Tie2-tmTNFα mice was characterized by increased expression of Jagged2 and by augmented Notch activation on hematopoietic stem/progenitor cells.

Conclusions

Our results provide the first evidence that BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch-dependent mechanism and that TNFα and LPS can modulate the levels of Notch ligand expression and Notch activation in the bone marrow microenvironment in vivo.

Introduction

In the adult, hematopoietic cells of all lineages arise from self-renewing stem cells and expanding progenitors embedded in the stromal fabric of the bone marrow (BM). The three-dimensional structure of the BM is constituted of the hematopoietic cells themselves, of extracellular matrix and of stromal cells, which include fibroblasts, adipocytes, osteoblasts and endothelial cells [1, 2]. Despite the increasingly recognized role of the BM endothelial cells in supporting hematopoietic multipotential progenitors, the molecular mechanisms involved and their potential implication in providing homeostatic stimuli to resident hematopoietic cells have been little investigated. As an interface between blood and tissue, the endothelium is poised to respond quickly to local changes elicited by trauma or inflammation [3]. This response both prevents physical disruption of the vessel wall by trauma, microbial organisms or toxins, and elicits host defenses. For instance, acute and chronic infections trigger the release of the pro-inflammatory cytokines IL-1β, IL-6, and TNFα that stimulate the host innate immune response. When stimulated by IL-1β or TNFα, BM endothelial cells (BM ECs) upregulate their cytokine production and adhesion molecules [4–6]. Physiologically, these events may facilitate the recruitment of inflammatory cells to contain the infection and, at the same time, impact on steady-state hematopoiesis such that progenitor’s expansion and differentiation may be modulated to respond to stress.

During embryogenesis, cell-cell contact between endothelium and hematopoietic cells is necessary for the development of the hematopoietic system [7, 8]. Similarly, in adult life the close association of BM endothelial cells with BM hematopoietic cells suggests that the endothelium continues to be a critical regulator of hematopoiesis [2, 9, 10]. BM ECs have an affinity for binding CD34+ progenitor cells [2, 11] and recent observations reported selective localization of purified hematopoietic stem cells (HSC) to the BM endothelium suggesting that BM endothelial cells could provide a specific functional niche for the HSC [12–14].

BM ECs constitutively produce cytokines that regulate expansion and differentiation of hematopoietic progenitors, such as interleukin-6 (IL-6), Kit-ligand, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Several studies have shown that BM EC monolayers are a unique type of endothelium that can support long-term proliferation of multilineage hematopoietic progenitors [2, 9, 15]. Although this effect has been largely attributed to cytokine production by BM ECs, cell-cell contact may play an important function that remains to be investigated. Cell-cell interactions between hematopoietic cells and their microenvironment play a critical role in the regulation of adult hematopoiesis. A class of molecules that govern cell differentiation and proliferation decisions through cell-cell contact is the Notch family of receptors and their ligands. Notch family members are highly conserved transmembrane receptors that play a critical role in regulating cell fate decisions in various organisms and in multiple tissues [16], including the hematopoietic tissues. Hematopoietic cells express Notch receptors and ligands and are surrounded, within the BM, by stromal cells expressing the Notch ligands Jagged1 (J1), Jagged2 (J2), Delta-like1 (Dll1) and Delta-like4 (Dll4) [17]. It is now well documented that the expression of Delta-like or Jagged family members on stromal feeder layers augment their ability to expand hematopoietic stem/progenitor cells in vitro [18–20]. Studies conducted by our group demonstrated that activation of Notch1 by J2 or Dll4 delays myeloid differentiation and preserves hematopoietic progenitors in a more immature phenotype [17, 21, 22]. Recently, J1 was found to be expressed by BM osteoblasts, a critical component of the BM stroma that elicits self-renewal of hematopoietic stem cells [23, 24]. Here, we show that endothelial cells of the BM microvasculature express the Notch ligand Jagged2 and that its expression is upregulated by inflammatory stimuli such as TNFα and LPS. We demonstrated that the effects of BM endothelium on BM progenitors is mediated by Notch signaling and depend on Notch-ligand density, thus contributing to a fine-tuned regulation of the stem/progenitor cells pool as it is needed during conditions of inflammatory stress. We hypothesized that BM endothelial cells may represent a functional niche that regulates homeostasis of the BM progenitor pool during inflammation.

Materials and Methods

Cells and cell cultures

BMEC cell line was generated as described [25] and provided by J. Ascensao and G. Almeida-Porada (University of Reno, Nevada). Primary human BM endothelial cells (BM-ECs) were isolated from BM of healthy donors (according to the guidelines of the Human Investigation Committee; DFCI) by purification with CD105 microbeads (Miltenyi) and selection in endothelial-supporting media, containing VEGF and bFGF (EGM-2 media; Cambrex). BMEC and primary BM-ECs were maintained with EGM2 medium and switched to DMEM 10% FBS 2 days prior and during TNFα or IL1β (Prepro Tech) stimulation. BM stroma from primary BM cells was generated using Dexter’s culture conditions as described [26]. Human CD34+ cells were derived from cord blood obtained from the Pediatric Research Institute, University of St. Louis, MO or the Saint Vincent’s Hospital, Indianapolis, IN (according to the guidelines of the Human Investigation Committee; IUSM) and isolated by immunomagnetic beads (Mylteni). Pools of CB CD34+ cells were grown in EX-VIVO-15 media (BioWHITTAKER), SCF (50ng/ml) and IL-3 (20ng/ml; R&D) in the presence or absence of BMEC. BMEC monolayers were formed in 24 well/plate or 6 well/plate wells in the presence of EGM2 media; 70–80% confluent monolayers were rinsed twice with IMDM media prior to coculture with CD34+ cells. CD34+ cells were seeded on BMEC monolayers at the concentration of 1×105 (on 24 well/plate wells) or 1×106 (on 6 well/plate wells), spinoculated for 30 minutes at 1500 RPM and cultured at 37°C and 5% CO2 for 1, 3 or 5 days. CD34+ cells and Notch antagonists. a) CD34+ and BMEC cocultures were grown for 5 days in the presence or absence of 2uM γ-secretase inhibitor GSI (GSI X, Calbiochem). b) Prior to seeding, CD34+ cells were blocked for 10 min on ice with human immunoglobulins (Sigma) and then incubated with saturating concentrations (25ug/ml) of soluble Dll4-Fc generated as described [22], the monoclonal antibody anti-N1 (Biomeda) [27, 28] or Fc fragment control (Sigma) for 1 hour on ice. The CD34+ fraction adherent to BMEC was harvested (using 1 rinse of PBS and 1 rinse of PBS/EDTA) and used for further analysis after 24 hours (for western blot analysis) or 3 days (for colony assay). Harvested CD34+ were seeded in methylcellulose at 3000 – 5000 cells/ml and evaluated for colony-forming ability at days 10–14 as described [21].

3T3 and MS5-Dll4 cell lines were generated by retroviral transduction as described [21]. BMEC-GFP and BMEC-J2 were generated by retroviral transduction with the MSCV-IRES-GFP vector alone or containing the full length cDNA of human Jagged2, following standard procedures [17, 21, 22].

Mice

Tie2-GFP reporter (FVB/N-TgN(TIE2GFP)287Sato), CD1 and C57Bl/6 mice were purchased from Jackson Labs. Hes5-GFP mice were BAC transgenics from the NIDS GENSAT project (Rockefeller University, www.gensat.org). Tie2-tmTNFα transgenic mice and WT non transgenic littermates were described in [29, 30]. Mice were matched for gender and analyzed at 4–5 weeks of age. rhTNFα (#410-MT) was from R&D Systems; Lipopolysaccharide (LPS) from Pseudomonas. aer. serotype 10 (#L8643) was from Sigma. 8–12 week old mice were each given 10 ug TNFα in PBS/0.1% BSA i.v. or 500 ug LPS in PBS i.p. and sacrificed at different time points. BM was flushed from two femurs from each mouse with PBS/0.2mM EDTA. All Animal Studies were approved by the MGH Subcommittee on Research Animal Care or by the Indiana University LARC Committee on Animal Research.

Immunological Reagents and Procedures

Human BM endothelial cells were harvested, blocked with human immunoglobulins and incubated for 30 min. on ice with the following antibodies: CD45, CD106 (VCAM), CD54-PE (ICAM-1) and CD144 (VE-Cadherin) from BD Pharmingen; CD105 from Invitrogen; AC133/2-PE, Neuropilin-PE (BDCA4) from Miltenyi Biotech; VEGFR 1/2/3 from R&D Systems; Von Willebrand (purified) from Serotec; CD144 (VE-Cadherin) from ; GaM-PE from BioSource. Murine BM mononuclear cells were flushed from femurs using PBS/EDTA (2mM). Prior to immunolabelling, cells were incubated with FC-receptor blocker (BD Pharmingen). BM cells were labeled with fluoroisothiocyanate- (FITC), allophycocianin- (APC) or percy-phycoerythrin-(PerCP) conjugated control immunoglobulins or specific monoclonal antibodies directed to: Sca1, c-Kit, CD31, FLK1, CD45, and the lineage markers cocktail (CD3, CD4, CD8, Gr1, CD19, NK). Intracellular staining was performed using fixing and permeabilization solutions from Caltag. Antibodies against J2, N1, N2, Val1744N1 or IgGs control (Jackson ImmunoResearch) were added at the concentration of 5 µg/ml for 30 min. Cells were washed and incubated with goat anti-rabbit antibody conjugated to phycoerythryn (PE) (Sigma) (1 µg/ml). Anti-J2 antibodies included the polyclonal antibody provided by J. Aster (Brigham and Woman Hospital[31], and the anti-J2 from Santa Cruz Biotechnology ( H-143). Anti-N1 antibodies included the polyclonal antibody provided by J. Aster [32], and the polyclonal from Santa Cruz Biotechnology (−20); anti-N2 was from SantaCruz (25–255). The antibody recognizing activated N1 was purchased from Calbiochem (Val 1744); anti-ICAM-1 (CD54) was from Becton Dickinson, San Jose, CA. Multicolor flow cytometric analysis was performed using the FACSCalibur instrument (Becton Dickinson, San Jose, CA).

Western blots were performed as described[33]. Antibody used for immunoblotting include anti-activated N1 (Calbiochem; Val 1744), anti N1 (C-20) and anti-β-actin (I-19) from SantaCruz. Signals were quantified using Molecular Dynamics scanner and ImageQuant analysis software. Murine femurs were fixed in zinc-fixative (BD Pharmigen) and decalcified by formic acid prior embedding in paraffin. BM sections were stained by using standard techniques with anti-J2 (H-143), anti-CD144 and anti-Flk1 antibodies (R&D) followed by donkey anti-rabbit Alexa 488 and by donkey anti-goat Alexa 647 (Molecular Probes). Images were collected on an Olympus FluoView IX2 confocal microscope using a 40X 1.3 NA oil immersion objectives and the appropriate filters for simultaneous detection of the Alexa 488 and Alexa 647 dyes. Several Z-sections collected at 0.62 um intervals were combined into single plane projections in Metamorph (Universal Imaging) cropping and minimal level adjustments were done in Adobe Photoshop.

Reverse transcription polymerase chain reaction and PCR (RT-PCR)

Total RNA was isolated using RNA Trizol (Invitrogen) and reverse transcribed with AMV reverse transcriptase (Boehringer Mannheim, Indianapolis, IN) and random examers primers (Boehringer Mannheim). The forward and reverse primers used for PCR are described in Table 1 of the supplemental information. PCR products were amplified through (30 cycles at 95°C for 60 seconds, 60°C for 90 seconds, and 72°C for 90 seconds on a GeneAmp 9600 termal cycler (Perkin Elmer, Roche Molecular Systems).

Statistical analysis

Equality of distributions for matched pairs of observations was tested using the T-Test.

Results

The Notch ligands Jagged are expressed by bone marrow endothelial cells

Given the respective roles of BM endothelium and of Notch signaling in maintaining and preserving multipotential hematopoietic progenitors in an immature state, we determined the expression profile of Notch ligands on BM endothelial cells and evaluated whether they may regulate hematopoiesis through stimulation of Notch signaling. As a model, we used the human BM-derived endothelial cell line BMEC, which has been immortalized by the SV40 large T-antigen, but still retains phenotypic and functional properties of endothelial cells [25]. In addition, we used primary human BM-derived endothelial cells (BM-ECs) derived from normal donors [34]. Immunophenotypic characterization of both BMEC and primary BM-ECs showed an endothelial-like phenotype. These cells are negative for CD45 and positive CD105 (Fig. 1A). CD45−CD105+ cells uniformly express: Neuropilin, VEGF-Rs, CD133, CD106, CD146, CD144, ICAM-1 and von Willebrand Factor, a combination of molecules that characterize endothelial cells (Fig. 1B–C). Furthermore, these BMEC and BM-ECs can form capillary networks, bind UEA-1, incorporate acetylated-LDL, inhibit smooth muscle cells, and respond to tumor stimuli with a signature similar to EC from other vascular beds [34, 35]. RT-PCR analysis of both BMEC cell line and primary BM-ECs showed expression of J1 and J2, but not detectable levels of Dll1 and Dll4 (Fig. 1D). The specific expression of J2 protein was further confirmed by flow cytometry analysis using antibodies that specially recognize J2 (Fig. 1E, left panel). Basal level of J2 expression was observed in the BMEC cell line (Fig. 1E, right panel) and in 5 out of 6 primary BM-EC samples analyzed (four cases showed in Fig. 1F), whereas it was not detectable in BM-derived stroma that lacked of the endothelial component (Fig. 1F).

Figure 1. J2 is expressed by BM-derived endothelial cells.

Figure 1

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(A–C) Characterization of human BM derived endothelial cells. BMEC clones (representing two different passages) and primary BM-ECs were harvested and labeled with the indicated antibodies in combination with CD45 and CD105 followed by multicolor analysis. (A) Dot blots show immunoglobulin controls (left) and expression of CD105 and CD45 in one representative sample. (B) CD45−CD105+ cells were analyzed for the expression of the indicated markers. Bar graphs represent 1 of 3 independent experiments. Numbers indicate average of mean fluorescence intensity of 3 samples for each antibody. Error bars represent standard deviation. (C) Histogram shows overlays of Neuropilin expression (dark gray filled curve) over IgG control (light gray filled curve) on: BMEC (left panel) and primary BM-ECs (right panel). (D) Notch ligand expression profile in human BM-derived endothelial cells. RNAs obtained from BMEC and BM-EC samples were used for RT-PCR amplification. All samples were positive for the low molecular size PCR product of the housekeeping gene GSα, confirming the absence of genomic DNA [22]. RNAs from MS-5 cells overexpressing human cDNA of each ligand were used as PCR positive control (PC). (E) Left panel: 3T3 cells were transduced with pBABE retrovirus alone or containing human J2 cDNA. Cells were selected by puromycin and J2 expression was confirmed by western blot analysis. 3T3-vector and 3T3-J2 cells were labeled with polyclonal anti-J2 antibodies followed by anti-rabbit PE-conjugated antibody. Histogram shows overlays of J2 expression on 3T3-vector (solid line) and on 3T3-J2 cells (dotted line); gray filled curve shows rabbit IgG control on 3T3-J2 cells. Right panel: Histogram shows overlays of J2 expression (solid line) and of control immunoglobulins (filled curve) on BMEC cells. (F) Flow cytometric analysis of J2 on primary BM-ECs. Histograms show fluorescence intensity on the x-axis and cell count on the y-axis. Superimposed are fluorograms with anti-J2 antibody (solid lines) and control immunoglobulins (filled curves) on BM-EC samples derived from four different donors and on BM stroma derived from a donor.

Upregulation of J2 on BM endothelial cells promotes expansion of hematopoietic progenitor cells by a Notch dependent mechanism

BM EC monolayers have a distinct ability to support long-term proliferation of multilineage hematopoietic progenitors [2, 9, 15], generally attributed to the production of hematopoietic cytokines. We investigated whether the ability of BM endothelial cells to maintain multipotential progenitors was also dependent on Notch activation through cell-cell contact and whether it correlated to the levels of J2 expression on BMEC. As anticipated, the colony-forming ability of human CD34+ progenitors was increased when co-cultured in the presence of BMEC cell line, compared to CD34+ progenitors cultured with recombinant cytokines alone (Fig. 2A). However, this effect was markedly reduced when ligand-dependent Notch signaling was inhibited by the γ-secretase inhibitor GSI X, which selectively blocks the protease-dependent cleavage of Notch in response to ligand binding [36]. As shown in Figure 2A, CD34+ cells co-cultured with BMEC cell monolayers in the presence of GSI generated a significantly lower number of colonies compared to CD34+ cells co-cultured with BMEC monolayers in the absence of GSI, indicating a critical role of Notch signaling in this process. Next, to determine the role of ligand density, we engineered BMEC cells overexpressing J2 (BMEC-J2). Exposure of CD34+ progenitors to increased ligand density on BMEC-J2 monolayers furthered their ability to form colonies compared to CD34+ progenitors co-cultured with BMEC cells control (BMEC-GFP; Fig. 2B). This effect was abrogated by inhibition of Notch signaling by GSI treatment and by the presence of a transwell (data not shown). Thus, cell-cell contact dependent Notch signaling is critical for the greater effect induced by BM-ECs on hematopoietic progenitors. Notch activation was confirmed by transcriptional activation of its target gene Hes1, and by the generation of the ligand-induced cleaved form of Notch (Notchic). Hes1 transcription was higher in cells cultured in the presence of BMEC cell lines (BMEC and BMEC-J2) than in cells cultured in their absence (no co-culture), and it was greatly reduced by GSI treatment (Fig. 2C). Accordingly, the cleaved activated form of Notch was visible only when CD34+ cells were co-cultured in direct contact with BMEC cells (Fig. 2D, lanes 2 and 3). To determine the specificity of this interaction, Notch-Jagged contact between CD34+ and BMEC cells was inhibited by blocking Notch receptors with an antibody directed to the extracellular domain of Notch1 (N1ab) [27, 28], or with the soluble form of Notch ligand Delta4 (Dll4-Fc) [22]. Both reagents can bind to N1 and N2; antibodies directed to ligand binding region of Notch receptors and soluble ligand-fusion proteins have been used as antagonist to inhibit the full physiologic ligand-receptor binding and activation [dontu27, 37–40]. Incubation of CD34+ cells with N1ab or Dll4-Fc prior seeding into BMEC monolayers diminished the robust Notch stimulation provided by the Jagged ligands on BMEC cells, as shown by the lower levels of cleaved Notchic generated in these conditions (Fig. 2D, left panel, lanes 3–5, and right panel). Similarly to the inhibition provided by GSI, decreased Notch signaling by pre-incubation with soluble Dll4-Fc or N1ab resulted in the decreased ability of cocultured CD34+ cells to generate colonies in a methylcellulose assay compared to CD34+ cells pre-incubated with control Fc (Fig. 2E), thus excluding potential indirect effects of GSI and confirming the specific role of Notch-ligand interactions.

Figure 2. BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch dependent mechanism.

Figure 2

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(A) CD34+ cells were grown in liquid culture on plastic alone in the presence of cytokines (CO) or on BMEC monolayers, in the absence or presence of GSI (2uM). After 5 days, cells were harvested, washed and plated in quadruplicate, at a density of 5,000 cells/ml, in methylcellulose supplemented with IL-3, SCF and EPO. Bar values indicate the average of 3 experiments. CFCs are expressed as number of colonies per 1000 cells seeded. Error bars represent standard error. Differences between the populations (BMEC vs CO; BMEC vs BMEC+GSI) are statistically significant p < 0.02. (B) CD34+ cells cocultured with BMEC-GFP or BMEC-J2 in the presence or absence of GSI (2uM) were harvested at day 5 of co-culture and plated in triplicate at the density of 5000 cells/ml in methylcellulose, as described above. Bars represent the average of 4 experiments. CFCs are expressed as number of colonies per 1000 cells seeded. Error bars represent standard error. Differences between populations (BMEC-J2 vs BMEC-GFP and BMEC-J2+GSI) are statistically significant p < 0.001; (C) RNAs from CD34+ cells cultured alone or in the presence of BMEC and BMEC-J2, in the presence or absence of GSI, were used for RT-PCR amplification of the Hes1 gene. The two blots are representative of two independent experiments. (D–E) CD34+ cells where incubated with Fc fragment (control), Dll4-Fc or anti-N1 antibody for one hour prior to seeding into BMEC monolayers. (D) Expression of activated Notch in CD34+ progenitors. CD34+ cells in the different conditions were harvested after 24 hours of coculture. Cell extracts were analyzed by immunoblot by using a Notch antibody detecting the activated form of Notch. Bar graphs represent ImageQuant densitometric analysis of Notchic protein level. Values indicate percentage of Notchic detected following incubation with N1ab or Dll4-Fc relative to the Fc control (100%). Notch densitometric values were normalized with β-actin levels. (E) CD34+ cells were harvested at 72 hours of co-culture and seeded at 3000/ml in methylcellulose. Bar graphs represent two independent experiments. Bars values are average of 4 wells and represents number of colonies, CFCs, per 1000 cells seeded. Error bars represent standard error.

Taken together, our results demonstrate that Notch signaling plays an important role in mediating the effect of BM endothelial on hematopoietic progenitor’s expansion and significantly potentiates the effects induced by hematopoietic cytokines produced by the BM endothelial cells. Furthermore, our data provide evidence that the regulation of ligand density on BM endothelial cells and Notch signal intensity on hematopoietic progenitors can result in different degrees of progenitor expansion.

TNFα upregulate J2 expression on BM endothelial cells in vitro and in vivo

Considering the importance of Notch ligand density on the cell surface, we asked whether microenvironmental cues could regulate their expression on BM endothelial cells. Since endothelial cells play a critical role in inflammation and their function is highly regulated by proinflammatory cytokines such as TNFα and Interleukin 1 β (IL1β), we determined whether J2 expression was modulated during cytokine-induced EC activation. The BMEC cell line and primary BM-EC cells were stimulated with TNF α (10ng/ml; optimal dose) for 24, 48 and 72 hours and ECs activation was confirmed by detection of IκB phosphorylation (data not shown) and upregulation of ICAM-1 (Fig. 3D). TNFα induced a significant upregulation of J2 expression in the BMEC cell line, both at the level of protein and transcript (Fig. 3A and B). Similarly, 3 out of 4 BM-EC samples upregulated J2 expression in response to TNFα by 24 hours (Fig. 3C). In all cases, J2 expression returned to basal levels by 48 hours. Of note, sample 4, which did not show detectable J2 expression at basal condition, responded with a strong upregulation of J2 following TNFα stimulation whereas no upregulation was observed in the BM stroma (Fig. 3C). Stimulation of BM endothelial cells with IL1β induced a weaker effect on J2 expression compared to TNFα as shown by a representative experiment in Fig. 3E.

Figure 3. J2 expression on BM-derived endothelial cells is upregulated by TNFα in vitro.

Figure 3

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(A) BMEC cell line was stimulated with TNFα (10 ng/ml), and cells harvested and labeled with anti-J2 antibody at the indicated time points. Histograms on the left show flow cytometric analysis of a representative experiment. Superimposed are fluorograms with anti-J2 antibody (solid line) and control immunoglobulin (filled curve). Bar graph on the right represents summary of multiple independent experiments (n=4). Numbers indicate average values of normalized Median Fluorescence Intensity (*MFI) of J2 expression relative to IgG controls (*MFI= MFI J2 - MFI IgG1). * Difference between populations is statistically significant: TNFα at 12 hrs vs 0 hrs p = <0.01. (B) RNAs obtained from BMEC samples stimulated with TNFα (10 ng/ml) were used for RT-PCR amplification of J2 and Gsα. (C) Primary BM-ECs were stimulated with TNFα (10 ng/ml), harvested and labeled with anti-J2 antibody. Histograms show intensity of J2 expression. Superimposed are fluorograms with anti-J2 antibody on cells stimulated with TNFα for 24 hours (solid lines) and 48 hours (dotted lines) and on cells not stimulated (basal expression, solid filled curve). (D) BMEC stimulated by TNFα (10 ng/ml) were harvested at the indicated time points and labeled with anti-CD54 (ICAM-1) monoclonal antibody. Histogram shows intensity of ICAM-1 expression. Superimposed are fluorograms with anti-ICAM-1 on cells stimulated with TNFα for 6 hours (solid thin line), 12 hours (overlap with the 6 hours) and 24 hours (dotted lines), and on cells not stimulated (solid thick line) and immunoglobulins control (solid filled curve). (E) The line graph shows a representative case of three (BM-EC1) stimulated with IL1β (10 ng/ml) or with TNFα (10ng/ml) for 3 days. Values in the graph represent values of normalized J2- *MFI during time.

Next, we confirmed the ability of TNFα to upregulate J2 expression on BM-ECs in vivo. CD1 or C57Bl/6 mice were injected with PBS or TNFα (10 ug) and their BM cells were analyzed by multicolor FACS analysis at 6, 12 and 24 hours after injection. J2 expression was evaluated on primary murine BM endothelial cells, identified by a combinatorial analysis of their surface markers as a population of large cells negative for CD45 and positive for both Flk1 (VEGFR2) and CD31 (Fig. 4A left panel). To further confirm the presence of endothelial cells within this population, we analyzed the expression of the endothelial cell marker TIE2. CD45− FLK1+CD31+ cells were highly enriched in endothelial cells co-expressing TIE2, as shown by analysis performed in TIE2-GFP reporter transgenic mice[41] (Fig. 4A, right panel), and showed high expression of ICAM1 and J2 by real-time PCR on sorted populations (data not shown).

Figure 4. TNFα and LPS upregulate J2 expression on BM endothelial cells in vivo.

Figure 4

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(A) Identification of endothelial cell population in the BM. BM cells from TIE2-GFP and not transgenic control mice were labeled with antibody directed to CD45, CD31 and FLK1. Dot blots on the left show expression of CD31 and FLK1 on gated CD45 negative cells (R2). Dot blots on the right show auto-fluorescence in the FL-1 channel and TIE2 promoter-driven GFP on gated CD45−CD31+FLK1+ population from a not transgenic mouse and a TIE2-GFP reporter mouse, respectively. CD31−/FLK1- cells (R3, bottom dot blot) were used as internal negative control for GFP. (B) Histograms show intensity of J2 expression on BM endothelial cells derived from mice injected with TNFα (10 ug) or LPS (500 ug) at 12 hours from injection compared to PBS. Superimposed are fluorograms with IgG control (IgG, solid filled curve, light gray) and anti-J2 antibody on cells derived from mice control treated with PBS (solid filled curve, dark gray) or stimulated with TNFα for 12 hours (solid line, black). (C) Bar graph represents summary of J2 upregulation by TNFα or LPS over time in multiple independent experiments (n = 4). Numbers indicate average of normalized MFI (*MFI) values for J2 expression on BM endothelial cells. Error bars represent standard deviation. * Difference between populations is statistically significant: TNFα vs PBS p = 0.03; LPS vs PBS p <0.01; (PBS n=10; TNF n=9; LPS n=5). (D) Bar graph represents average values of TNFα (pg/ml) in the serum of control mice or mice injected with LPS, at 12 hours. Serum was collected from 3 animals/group and triplicate samples for each mouse were analyzed by ELISA. Error bars represent standard deviation. * Difference between populations is statistically significant: p = 0.0012.

TNFα administration resulted in a 2 to 3 fold increase of J2 expression in BM-ECs over the baseline (Fig. 4B and C). Time course analysis showed that J2 upregulation was at its highest at 12 hours from TNFα injection and declined over time (Fig. 4C). Overall, these results confirm the ability of TNFα of upregulating J2 expression on BM-ECs in vivo and provide evidence for the modulation of Notch ligand expression by microenvironmental cues.

To investigate the physiological relevance of these findings, we analyzed the expression of J2 on BM-ECs following lipopolysaccharide (LPS) inoculation in vivo. Bacterial LPS, following bacterial infection, are the major triggers of TNFα release and of inflammatory response. LPS bind to Toll-like receptor 4 (TLR4) on endothelial cells resulting in their activation and intense production of TNFα during bacterial infection [42]. In our experiments, in vivo induction of TNFα by LPS was confirmed by evaluation of TNFα serum levels by ELISA after LPS inoculation (Fig. 4D). Similarly to TNFα injection, LPS challenge resulted in a dramatic upregulation of J2 expression on BM-ECs, highest at 12 hours from the injection (Fig. 4B and C), suggesting that J2 upregulation occurs in response to bacterial infection.

TNF and LPS stimulation result in increased expression and activation of Notch in BM progenitors

Next, we examined the expression and activation status of Notch on hematopoietic progenitors in response to TNFα stimulation. Both TNFα and LPS injection resulted in approximately 2 fold upregulation of N1 expression on Lin−Sca+ cells (Fig. 5A and B). N1 expression was analyzed on the larger population of Lin−Sca+ cells to allow a statistical significant number of events to be measured; however a similar trend was observed in the smaller Lin−Sca+Kit+ subset. Treatment with TNFα resulted also in an increase of N2 expression (Fig. 5C).

Figure 5. TNF and LPS modulate N1 expression and activation on BM progenitors in vivo.

Figure 5

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(A–F) BM cells were harvested from mice inoculated with PBS, TNF (10 ug) or LPS (500 ug) at 12 hours from injection and were labeled with antibodies directed to lineage markers (Lin), Sca1, c-Kit and than with anti-N1 antibody or antiN1Val1744. (A) Histograms show N1 expression on gated Lin−Sca1+ cells, as shown in the dot blot on the left panel. Superimposed are fluorograms with IgG control (solid filled curve) and with anti-N1 antibody (solid line, black) on cells derived from mice inoculated with PBS (controls) or stimulated with either TNFα or LPS. (B) Bar graph represents summary of N1 expression on Lin−Sca1+ cells in multiple independent experiments (n = 4). Values represent average of fold increase of N1 expression (*MFI) in mice that showed upregulation. Number in parenthesis indicates the total number of mice analyzed. Error bars represent standard deviation. To avoid bias due to random variation of fluorescence of intensity, each MFI value for N1 was normalized toward its IgG control (*MFI); in each experiment, the ratio between N1 *MFI in animals challenged with TNFα or LPS and N1 *MFI in mice that received PBS was calculated and expressed as fold difference. (C) Bar graph represents summary of N2 expression on Lin−Sca1+ cells. Values represent average of *MFI in control or TNFα challenged mice (n = 4). Error bars represent standard deviation. (D) Lin−Sca+ gated BM cells were analyzed for expression of activated N1 using the monoclonal antibody against N1ic (Val 1744). Superimposed are fluorograms with N1ic (Val 1744) antibody on cells harvested from TNFα-stimulated (solid line) or control mice (filled curve). (E) Bar graph represents summary of N1ic (Val 1744) expression on Lin−Sca1+ cells in independent experiments (n = 4). For each condition, values represent average of mean intensity of fluorescence of samples labeled with IgG control (gray bars) or with N Val1744 antibody (black bars). (F) Hes5-GFP transgenic mice were injected with PBS or LPS. BM cells were harvested 12 hours after injection, labeled with the indicated antibodies (x-axis) and analyzed by flow cytometry for expression of GFP. Graph shows average percent GFP expression in Lin−Sca1+Kit+ and Lin−Sca1+ subsets. Error bars represent standard deviation.

More importantly, increased expression of J2 and of N1 and N2 following TNFα or LPS injection, was accompanied by increased Notch activation, as determined by the levels of the active form of Notch1 (Notchic) on Lin−Sca+ cells detected by an antibody that specifically recognizes the cleaved intracellular form of Notch generated upon ligand binding [22] (Fig. 5D and E).

To complement this analysis and further demonstrate the activation of Notch signaling during inflammation in vivo, we injected LPS into transgenic mice expressing GFP under the control of the endogenous Hes5 promoter. Hes5 transcription is tightly and directly activated by Notch signaling in vivo [43]. Basal levels of HES5-driven GFP expression were barely detectable by flow cytometry in the BM cells of transgenic animals at steady-state conditions. In contrast, LPS challenge induced a significantly increase in Hes5-driven GFP expression in both Lin−Sca+ and Lin−Sca+Kit+ subsets (Fig. 5F).

In conclusion, these results provide evidence that pro-inflammatory stimuli, such as TNFα and LPS, result in the activation of Notch signaling on hematopoietic progenitors in vivo.

Upregulation of Notch signaling in the bone marrow microenvironment of Tie2-tmTNFα mice

Systemic administration of recombinant TNFα in vivo affects directly and indirectly multiple cellular types. To further corroborate the results obtained with soluble TNFα, we analyzed the levels of J2 expression and Notch signaling activation in a model of endothelial cell activation by using Tie2-tmTNFα transgenic mice. In Tie2-tmTNFα transgenic mouse, a transmembrane, noncleavable form [mTNFD1–9,K(11)E] of TNFα is expressed under the control of the Tie2 promoter, in conjunction with an intronic enhancer element used to drive endothelial-specific expression [30]. In these mice, endothelial cells exhibit a chronic activated phenotype as result of constitutive expression of the TNFα membrane-bound form [29, 30]. Analysis of J2 expression in Tie2-tmTNFα mice showed increased levels of J2 on BM cells with endothelial immunophenotype CD45−CD31+FLK1+, compared to non-transgenic (WT) controls (Fig. 6A and B). In both, WT and Tie2-tmTNFα expression of J2 was specific for CD45−CD31+FLK1+ cells and was absent in the CD45−CD31+FLK1− population (Fig. 6A). Similarly to what observed during recombinant TNFα injection, increased levels of J2 on Tie2-tmTNFα BM-ECs were associated with increased expression of Notchic on Lin−Sca+Kit+ progenitors (Fig. 6C). As physiologic Notch signaling can be activated only by its cognate ligand, these results suggest a direct link between augmented J2 on BMEC cells and Notch activation on hematopoietic progenitors. Finally, given the robust expression of J2 in the BM-ECs of Tie2-tmTNFα transgenic animals (Fig. 6A–B), we utilized this model to demonstrate endothelial J2 expression in the BM based on its co-localization with VE-Cadherin/ Flk1 expression by confocal microscopy (Fig. 6D).

Figure 6. Jagged2 and Notch signaling are upregulatated in TNFαtm mice bone marrow.

Figure 6

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BM cells from Tie2-tmTNFα and not transgenic control mice were labeled with antibody combination directed to CD45, CD31 and Flk1, and directed to lineage markers (Lin), Sca1, c-Kit. (A) J2 expression on CD45−CD31+Flk1+ BM cells. CD45/CD31/Flk1 staining was followed by intracytoplasmic labeling of J2. Histograms show intensity of J2 expression on gated populations of non-transgenic and Tie2-tmTNFα mice. Superimposed are fluorograms with anti-J2 antibody on CD45−CD31+Flk1+ cells (solid filled curve, dark gray) and anti-J2 antibody on CD45−CD31+Flk1− cells (solid filled curve, light gray); anti-J2 antibody fluorescence on CD45−CD31+Flk1− coincided with IgG controls (not shown). (B) Bar graph represents summary of J2 upregulation in CD45−CD31+Flk1+ cells from non transgenic and Tie2-tmTNFα mice (n = 6). Numbers indicate average of normalized MFI (*MFI) values for J2 expression on BM endothelial cells. Error bars represent standard deviation. * Difference between populations is statistically significant: p < 0.01. (C) Notchic expression on LSK cells. Lin/Sca1/c-Kit staining was followed by intracytoplasmic labeling of cleaved Notch using the monoclonal antibody against N1ic. Bar graph represents summary of Notchic upregulation in Lin−Sca+c-Kit+ cells from non transgenic and Tie2-tmTNFα mice (n = 6). Numbers indicate average of normalized MFI (*MFI) values for Notchic expression on LSK cells. Error bars represent standard deviation. * Difference between populations is statistically significant: p = 0.01. (D) Co-localization of J2 and endothelial markers on endothelial cells of BM microvasculature. Images show a microvessel within the BM of a Tie2-tmTNFα mouse. Fixed and decalcified BM sections were stained with rabbit anti-J2 and a mixture of goat anti-VE-cadherin and goat anti-Flk1 antibodies. Donkey anti-rabbit antibody conjugated with Alexa 488 and donkey anti-goat antibody conjugated with Alexa 647 were used as secondary antibody. BM sections were imaged by an Olympus FluoView IX2 confocal microscope. Images show: 40 × magnification of the BM section stained with J2 antibody (green; far left panel); 80 × magnification of BM section with J2 (second panel, green) and VE-Cadherin+ Flk1 staining (red; third panel), merging in an endothelial cell lining in a BM microvessel (yellow; far right panel).

Discussion

The results described here, provide the first demonstration that primary BM endothelial cells express the Jagged family of Notch ligands and that such expression can be regulated by TNF α and LPS in the bone marrow in vivo. Furthermore, we showed that the distinct ability of BM endothelial cells to support hematopoietic multipotential progenitor cells in vitro, is not exclusively related to cytokines production but is also mediated by a Notch-mediated mechanism. Our results demonstrate that BM endothelial cells express the Notch ligand J2 and promote a greater expansion of hematopoietic progenitor cells through a Notch-mediated mechanism.

It is known that Notch signaling by itself can not bypass growth factor requirement, particularly in primary cells, and necessitates the presence of serum or cytokines to manifest its effects [22]. Indeed, there are many evidences that Notch signaling and hematopoietic cytokines synergize in promoting multipotential progenitor expansion. Independent studies have demonstrated synergism between Notch signaling and various hematopoietic cytokines (i.e. SCF, IL-6, IL-11, Flt-3l, IL7, IL-3 and TPO) in promoting multipotential progenitor expansion [44, 45]. BMEC cells express hematopoietic cytokines (GM, SCF, IL6) [25] and express Notch ligands. These two functions could be distinguished in vitro through inhibition of Notch-Jagged binding by Notch antagonists, such the γ-secretase inhibitor, soluble Dll4-Fc or anti-Notch monoclonal antibody, which significantly decreased the capability of BMEC of promoting CD34+ cell colony-forming ability and revealed the role of cytokines alone. We have also demonstrated that the higher the expression of J2 on BMEC cells, the higher is their efficiency of supporting hematopoietic progenitors. As shown by a recent study, Notch-ligand density upregulation by as much as 2-fold has quantitative and qualitative impact on hematopoietic progenitor cell differentiation and proliferation [46].

Thus, it is relevant to investigate whether microenvironmental cues could regulate Notch ligands expression on endothelial cells. We found that known regulators of endothelial cell activation, such as TNFα, ant at less extend IL1-β, upregulate J2 expression on BM endothelial cells. In this perspective, activation of BM endothelial cells by TNFα has a potent effect, resulting both in the induction of hematopoietic cytokines [4–6] as well as in the upregulation of Notch ligands, which combined can promote more efficiently progenitor expansion than cytokines alone [44, 45]. Furthermore, we provide evidence that J2 expression on BM endothelial cells can be upregulated 3-to 4 fold by TNFα and LPS in vivo. Overall, our findings link for the first time Notch as an intrinsic regulator of hematopoiesis, with TNFα, a critical regulator of inflammation, and suggest a partnership between Notch signaling and BM endothelial cells in the regulation of hematopoiesis during inflammation.

A role for Notch in inflammation is supported by previous studies showing the interplay between Notch signaling and the TNFα/NF-κB pathway. Overexpression of the NF-κB members c-Rel or p65 has been shown to induce J1 expression on HELA cells, whereas a NF-κB dominant negative form suppresses it [47]. Similarly, another group reported the upregulation of N1, N4 and J2 by TNFα on rheumatoid synovial fibroblasts [48]. On the other hand, numerous in vitro and in vivo studies have demonstrated the ability of Notch to expand stem/multipotential progenitors [49]. The role of the osteoblastic stem cell niche in supporting hematopoiesis through Jagged1 has been shown by Calvi et al. [23, 24]. However, the observation that BM endothelial cells are also in tight contact with hematopoietic stem/progenitor cells [12] poses the possibility that multiple stem cell niches exists in the BM, sensing different microenvironmental cues and providing different functions. Since BM ECs are finely regulated by inflammatory stimuli, our results suggest the potential role of the “endothelial stem cell niche” to regulate stem/progenitor cells homeostasis during condition of inflammatory stress. Stimulation of endogenous Notch with the physiologic ligands delays myeloid differentiation and preserves hematopoietic progenitors in a more immature phenotype [18, 19, 21, 50, 51]; moreover activation of Notch in murine stem cells promotes stem cell expansion in vivo [52]. However, conditional knock-out mice of N1, N2 or J1 do not exhibit significant alterations in the stem cells and progenitor compartment [53, 54], suggesting compensatory mechanisms by the other Notch receptors and by the other Notch-ligands, and perhaps a limited role of Notch signaling in stem cells regulation at steady-state conditions. Indeed, many studies have shown that in vitro exposure of hematopoietic stem/progenitor cells to Notch ligands promote their expansion while opposing to their differentiation [18–21], indicating that hyperactivation of Notch signaling may play a physiologic role in response to conditions of stress.

The physiologic relevance of our findings is confirmed by the demonstration that TNFα or LPS, which is a major trigger to bacterial response, upregulate J2 expression on BM endothelial cells in vivo and correlate with increased Notch activation. Of note, J2 upregulation appeared to be more robust when induced by LPS than when induced by TNFα alone. One explanation for this effect is that LPS stimulation may induce a more constant release of TNFα in vivo or/and that TLR4 activation by LPS may directly contribute to J2 regulation, as shown recently for the induction of Dll4 by LPS in macrophages [55]; however further studies are required to assess the distinct role of Toll receptors in modulating Notch signaling in hematopoietic cells during infections. In response to inflammatory stimuli, Notch1 and Notch2 expression was increased on hematopoietic cells, likely due to a direct effect of TNFα [48]. Expression of Notch receptors do not correlate necessary with their activation status, which is dependent on the presence and abundance of the ligand on neighboring cells. Thus, it is most relevant our observation that J2 upregulation on BM endothelial cells is associated with the increase of Notch activation on hematopoietic progenitor cells following TNFα or LPS stimulation. Since TNFα and LPS do not have a direct effect on Notch-receptor intracytoplasmic cleavage and activation, which can occur only upon binding to a Notch-ligand ligand on a neighboring cells, our data suggest the existence of endothelial-hematopoietic cell-cell interaction similar to that we have observed in the co-culture model in vitro. This notion is further supported by analysis of the Tie2-tmTNFα mice. In these mice, endothelial cells exhibit a chronic activated phenotype as result of endothelial-specific constitutive expression of the TNFα membrane-bound form [29, 30] and not in response to exogenous TNFα. As a result of endothelial cell activation, we found that J2 expression was upregulated in BM endothelial cells and that it correlated with increased Notch activation on Lin−Sca+Kit+ hematopoietic progenitors.

Increased of Jagged may have an impact on endothelial cells themselves following TNFα stimulation. Studies conducted during mouse development showed an inhibitory effect of Dll4 on arterial formation [56]. We have investigated the effects of increased Notch activation on BMEC cells by overexpressing the Notch ligands (Carlesso, unpublished observations). Although ligands overexpression increased Notch signaling in BMEC cells and resulted in some alteration of their proliferation, we did not observe inhibitory effects on co-cultured hematopoietic cells, but rather stimulatory effects as the ligand density on the BMEC surface was higher. Further in vivo studies are necessary to determine the vascular biology of adult BM endothelial cells during inflammation.

Overall, our data suggest that conditions associated with TNFα secretion and endothelial cell activation, such as infection and inflammation, result in increased endothelial cytokine secretion and increased Notch-ligand density on their surface on one hand, and in the increase in Notch signaling on hematopoietic progenitors on the other hand. Given the proximity of endothelium and hematopoietic progenitors in the BM, it is conceivable that the endothelial niche plays a role in hematopoietic progenitor activation and expansion in vivo, in particular under conditions of inflammatory stress. The in vitro co-culture experiments of BM-ECs and hematopoietic cells and the in vivo studies demonstrating selective localization of hematopoietic stem cells to the BM endothelium [14], support this view; however, further studies implying genetic models and in vivo imaging will be necessary to demonstrate in vivo the specific Notch-ligand interactions between BM endothelium and stem cells.

In conclusion, we propose a working model in which the BM endothelium represents a functional niche capable of regulating progenitor’s homeostasis in response to inflammatory stimuli through a mechanism that involves Notch signaling.

Acknowledgments

This work was supported by NIH R01 HL68256-01 (N.C.), the Foundation for Science and Technology, Portugal (FCT), The Harvard 50th Anniversary Program for Scholars - Claflin Distinguish Scholar Award and Nesson Award (N.C.), FCT research grant (A.A.C.), and FCT graduate program scholarship (A.C.).

We thank the precious technical assistance and intellectual contribution of Jaquenelson Fernandes and Miriam Conces; Graca Almeida-Porada for providing the BMEC cell line and Jon Aster for providing anti-J2 and anti-N1 antibodies.

Footnotes

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