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The Journal of Immunology, 2002, 169: 4273-4278.
Copyright © 2002 by The American Association of Immunologists

The Notch Ligand Jagged-1 Is Able to Induce Maturation of Monocyte-Derived Human Dendritic Cells1

Sanne Weijzen*, Markwin P. Velders1,*, Amira G. Elmishad*, Patricia E. Bacon{dagger}, Jeffrey R. Panella{dagger}, Brian J. Nickoloff{dagger}, Lucio Miele{ddagger} and W. Martin Kast2,*

* Cancer Immunology and {dagger} Skin Cancer Programs, Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL 60153; and {ddagger} Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Notch receptors play a key role in several cellular processes including differentiation, proliferation, and apoptosis. This study investigated whether the activation of Notch signaling would affect the maturation of dendritic cells (DCs). Direct stimulation of Notch signaling in DCs with a peptide ligand induced DC maturation, similar to LPS: DCs up-regulated maturation markers, produced IL-12, lost endocytosis capacity, and became able to activate allogeneic T cells. Furthermore, coculture of DCs with cells expressing Notch ligand Jagged-1 induced up-regulation of maturation markers, IL-12 production, T cell proliferative responses, and IFN-{gamma} production. Our data suggest that activation of Notch by Jagged-1 plays an important role in maturation of human DCs. Additionally, they reveal a novel role for Notch signaling in cell maturation events distal to the cell fate decision fork. These data may have important medical implications, since they provide new reagents to induce DC activity, which may be beneficial as adjuvants in situations where an immune response needs to be elicited, such as tumor immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DCs)3 are a family of professional APCs that play a key role in the induction of T cell responses. Many subsets of DCs have been identified in both mice and humans (i.e., plasmatoid DCs, lymphoid DCs, myeloid DCs) (1, 2, 3, 4) All these subsets undergo a similar developmental differentiation program, called DC maturation. Immature DCs are mostly located in peripheral tissues and are highly efficient at Ag uptake by phagocytosis, receptor-mediated endocytosis, and macropinocytosis. After Ag uptake, DCs mature, become specialized in Ag presentation, and migrate to local lymph nodes to initiate immune responses. Maturation can be induced by diverse signals, such as tissue damage or infection. It can be triggered by components of the bacterial wall (i.e., LPS), inflammatory cytokines (TNF-{alpha} or IL-1), or viral components (i.e., dsRNA, virus-like particle) (see review in Ref. 5). Signal transduction events following maturation signals depend largely on the specific signaling receptor that is activated (TNFR, Toll-like receptors (TLR)), but all signals seem to route primarily through NF-{kappa}B (6).

Notch receptors are a family of highly conserved heterodimeric transmembrane proteins involved in various cell fate decisions, such as proliferation, differentiation, and apoptosis (7). Mammals have four known Notch receptors, which vary in tissue and cellular distributions. Activation of the Notch pathway is triggered by binding of ligands that are generally cell membrane associated. Engagement of the receptor induces cleavage of the transmembrane subunit (NTM), releasing an intracellular domain (NIC) that translocates to the nucleus. Nuclear NIC exerts transcriptional control of its many target genes, including basic helix-loop-helix transcription factors, primarily through the transcriptional regulator C promoter binding factor-1/recombination signal sequence binding protein-Jk (8). Notch signaling regulates differentiation of numerous cell types (9, 10, 11, 12) Among cells of the hemopoietic lineage, Notch mRNA and protein expression can be detected in human immature CD34+ hemopoietic progenitors; lymphoid, myeloid, and erythroid precursors; as well as B and T cells, monocytes, and neutrophils (13, 14). Notch signaling is involved in lineage decisions and differentiation at several stages during hemopoietic development (15, 16, 17) One study reported that inducible Notch-1-deficient mice have normal DC development. Neither peripheral DCs nor Langerhans cells were affected by deficient Notch-1 signaling (18). However, that study did not address the functionality of the Notch-1-deficient DCs and only investigated immature DCs. Therefore, we investigated whether activation of Notch-1 signaling would influence the maturation of human monocyte-derived dendritic cells. We show that direct engagement of the Notch receptor by a soluble Notch ligand or native Jagged-1 naturally expressed on primary keratinocytes or transfected into Jagged-negative fibroblasts induces phenotypical and functional DC maturation. These data indicate that activation of Notch-1 signaling by Jagged-1 can induce maturation of human DC. Moreover, they identify a new class of reagents that can mature DCs in vitro through the Notch signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and plasmids

Human DCs were generated from PBL obtained from healthy donors by leukapheresis. Leukocytes were purified by Lymphoprep (Nycomed, Oslo, Norway) and were stored until further use. Frozen PBL were thawed and washed in RPMI 1640 containing 2 mM glutamine (Mediatech, Herndon, VA) 10 mM pyruvic acid (Life Technologies, Gaithersburg, MD), 10 mM nonessential amino acids (Life Technologies), 100 µg/ml kanamycin (Sigma-Aldrich, St. Louis, MO), 10% FCS (HyClone Laboratories, Logan UT), and plastic-adherent cells were selected after incubation for 2 h at 37°C in 5% CO2. Nonadherent cells were washed away, and adherent cells were cultured for 3 days in 1000 U/ml recombinant human GM-CSF (Intergen, Purchase, NY) and 1000 U/ml recombinant human IL-4 (Intergen). On day 3 nonadherent cells were collected, washed in PBS, and incubated for 1 h in 1 ml in the presence of one of the following activation stimuli: 10 µg/ml LPS (Sigma-Aldrich), TNF-{alpha} (PeproTech, Rocky Hill, NJ), 50 µM Jagged peptide, 50 µM Scrambled peptide, human keratinocytes, fibroblasts transfected with human Jagged-1 or empty vector, or PBS alone as a negative control. DCs were then cultured for an additional 48 h before use in phenotypical or functional assays. In the case of coculture with keratinocytes or fibroblasts, DCs were gently collected after 48 h, leaving the monolayer of keratinocytes or fibroblasts intact. This guaranteed a minimal contamination of DCs with keratinocytes or fibroblasts. To ensure that the expression of measured activation markers was not the result of contamination, the expression of MHC class I or II, CD80, CD83, CD86, CD40, and CCR7 on keratinocytes and fibroblasts alone was determined.

Normal human keratinocytes were isolated from neonatal foreskins and grown in low calcium (0.07 mM), serum-free medium (Clonetics, San Diego, CA) on plastic dishes as previously described (19). Normal fibroblasts were isolated from a 4-mm skin biopsy that was incubated overnight at 4°C in dispase II (Roche, Indianapolis, IN) and RPMI 1640 (Life Technologies). Dermis and epidermis were separated, dermis was minced, and the small pieces were incubated in trypsin/EDTA (Life Technologies) for 4 h at 37°C, after which IMDM containing 10% FCS was added. Human Jagged-1 cDNA was provided by Dr. S. Artavanis-Tsakonas (Harvard Medical School, Boston, MA) and subcloned into NotI in pLZRS. The Phoenix-Ampho packaging cell line (American Type Culture Collection, Manassas, VA, with permission of Dr. G. Nolan, Stanford University Medical Center, Stanford, CA) was transfected as previously described (20). Fibroblasts were infected with Jagged or empty vector as previously described (20).

IL-12 and IFN-{gamma} ELISA

For IL-12 ELISA, 1.5 x 106 day 3 DCs were incubated in the presence of the appropriate maturation stimulus in a six-well plate. After 24 h, supernatant was collected, and the amount of IL-12 was determined with an IL-12(p70) ELISA (Endogen, Woburn, MA). IFN-{gamma} production was measured in supernatants collected from MLR on day 4. IFN-{gamma}-specific, ELISA-matched Abs were used.

Western blot

Immunoblotting of cellular extracts was performed after SDS-PAGE on 7 or 10% Tris-acetate gels (Invitrogen, Carlsbad, CA). Equal protein loading was verified by Bradford protein assay and by stripping and reprobing membranes with anti-GAPDH.

Abs and reagents

Abs to human Notch-1, Jagged-1 (Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (Chemicon, Temecula, CA) and secondary Abs anti-goat IgG-HRP (Vector, Burlingame, CA) and anti-mouse/rabbit F(ab)2 IgG-HRP (Roche, Mannheim, Germany) were used for Western blotting. Abs to human CCR7, CD40, CD80-FITC, CD86-FITC, CD83, isotype controls, streptavidin-allophycocyanin, and HLA-DR, -DQ, and -DP-FITC were purchased from BD PharMingen (San Diego, CA); goat anti-mouse FITC was purchased from BioSource International (Camarillo, CA); and HLA-A-, -B-, and -C-PE was obtained from DAKO (Glostrup, Denmark). Jagged (aa 188–204; CDDYYYGFGCNKFCRPR) and Scrambled (RCGPDCFDNYGRYKYCF) peptides were synthesized at the University of Chicago Chemical Core facility. An endotoxin test (Sigma-Aldrich) showed that these reagents contained <0.015 endotoxin units/ml, the lowest standard in the kit.

Mixed leukocyte reactions

DCs (1 x 103) were cocultured with 1 x 105 allogeneic PBL in a 96-well plate. As a positive control for stimulation of T cells, Staphylococcus enterotoxin B (Sigma-Aldrich) was added to PBL. Four days after the start of the culture, 100 µl supernatant was replaced by 1 µCi [3H]thymidine/well. After 18 h, cells were harvested onto filter plates (Packard Instrument, Meriden, CT), and scintillation fluid was added (Microscint-20; Packard Instrument). 3H incorporation was determined using a TopCount scintillation counter (Packard Instrument).

Statistical analysis

The effects of Jagged peptide or Jagged-1 expressed on keratinocytes or fibroblasts on DC maturation compared with immature DCs were analyzed by two-tailed t test with a significance level of 0.05, using GraphStat software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DCs gain a mature phenotype after direct stimulation of the Notch signaling pathway

To determine whether activation of Notch-1 in DCs in the absence of a known DC maturation signal results in DC maturation, Notch signaling was stimulated in immature DCs in the absence of LPS. For this purpose, a synthetic peptide (Jagged), corresponding to the {Delta}/Serrate/LAG-2 domain of hJagged1, was used. The 17-mer peptide binds to the extracellular portion of the Notch receptor and initiates the signaling cascade (21). As a negative control, a synthetic peptide (Scrambled) with the scrambled sequence of the Jagged peptide was used (22). These reagents were tested for the presence of endotoxin to rule out DC activation as a result of endotoxin contamination. The concentration of endotoxin was <0.015 endotoxin units/ml, which is a concentration of endotoxin far below the level needed for DC activation (23). Exposure of immature DCs to Jagged peptide at the time when they are normally exposed to LPS resulted in appearance of active Notch (NIC), in contrast to exposure of DCs to Scrambled or control DCs (Fig. 1GoA).



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  		FIGURE 1. Direct stimulation of the Notch receptor leads to a mature DC phenotype in human DCs. DCs were cocultured with a synthetic Jagged peptide, which is able to activate the Notch signaling pathway. A, The functionality of this peptide was determined by Western blot analysis of DCs treated with Jagged or Scrambled peptide. A representative Western blot is shown of four performed. Notch protein was determined 48 h after the start of treatment. B, Phenotypical analysis of DCs cocultured with the Jagged or Scrambled peptide was performed by FACS, where the expression of MHC class I or II, CD80, CD86, and CD83 was compared with LPS-matured DCs. The dark gray line represents expression in immature DCs (control and Scrambled overlap), the light gray line represents Jagged-treated DCs, and the black line represents LPS-matured DCs. A representative FACS analysis is shown of 10 performed. C, The ability to endocytose Ag was determined using self-quenching fluorescent BSA. Upon uptake and degradation of BSA, fluorescence is dequenced. Immature or LPS-, Jagged-, or Scrambled-treated DCs were analyzed for FITC and CD83 expression by FACS. A representative FACS analysis is shown of three performed.

 
To assess maturation of DCs by Jagged, the expression of maturation markers was determined by flow cytometry (Fig. 1GoB). A significant up-regulation of MHC class I and II, CD80, CD83, and CD86 surface expression on Jagged-treated DCs was detected. DCs exposed to LPS showed similar expression levels as DCs treated with Jagged. Incubation of DCs with Scrambled peptide did not affect the expression levels of these maturation markers, which were similar to those of immature DCs. The level of up-regulation of maturation markers after Jagged treatment was equal to or higher than up-regulation measured after treatment with CD40L (0.5 µg/ml; data not shown).

An important property of immature DCs is their ability to internalize and process Ags. To examine Ag uptake by DCs after exposure to Jagged, DCs were incubated with self-quenching dye conjugates of BSA that gains green fluorescence only upon internalization and subsequent degradation. While control or Scrambled-treated, immature DCs rapidly internalized and degraded BSA at 37°C, as indicated by a high fluorescent signal, neither LPS-treated nor Jagged-treated DCs internalized the green fluorescent BSA. The maturation status of these DCs was confirmed by high expression of CD83, which was low in the immature DCs (Fig. 1GoC).

DCs matured by direct stimulation of Notch are functionally active

Up-regulation of maturation markers after stimulation of the Notch receptor is an indication that immature DCs have differentiated into their mature form. To determine whether these Jagged-treated DCs are also functionally active, the production of bioactive IL-12(p70) (24, 25) and T cell activation was determined. Both LPS- and Jagged-treated DCs produced IL-12(p70), whereas immature DCs and Scrambled-treated DCs did not (control vs Jagged, p < 0.05). However, IL-12(p70) production by LPS-treated DCs was significantly higher than that in Jagged-treated DCs (Fig. 2GoA).



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  		FIGURE 2. Jagged-treated DCs are functionally mature. Human DCs were cultured in the presence of 50 µM Jagged or Scrambled peptide for 48 h. The functionality of the DCs cultured with Jagged was compared with that of DCs cultured with LPS. Representative experiments are shown of three performed. A, IL-12 (p70) ELISA was performed 24 h after the start of culture with LPS or Jagged. B, An MLR was performed using DCs cultured in the presence of LPS, Jagged, or Scrambled as stimulators and allogeneic PBL as responders. [3H]thymidine incorporation was determined as a measure for T cell proliferation. C, IFN-{gamma} production was determined by ELISA in an MLR using DCs cultured in the presence of LPS, Jagged, or Scrambled as stimulators and allogeneic PBL as responders.

 
To establish T cell activation, differently treated DCs were cocultured with allogeneic PBL in an MLR. After 4 days, supernatant was collected for cytokine analysis, and [3H]thymidine was added to measure the proliferation of PBL. DCs matured by LPS or Jagged induced high levels of T cell proliferation (control vs Jagged, p < 0.001; Fig. 2GoB). The proliferation levels observed were comparable to those induced by Staphylococcus enterotoxin B. Immature or Scrambled-treated DCs induced similar low T cell proliferation. DCs or T cells alone did not proliferate measurably. IFN-{gamma} production is another indicator of T cell activation. Significant IFN-{gamma} production by T cells was measured by ELISA in response to LPS- or Jagged-stimulated DCs (control vs Jagged, p < 0.01). Stimulation of PBL by immature DCs or DCs exposed to Scrambled peptide induced a significantly lower IFN-{gamma} production (Fig. 2GoC). In summary, these data show that DCs stimulated through the Notch signaling pathway, not only exhibit a mature DC phenotype, but are functionally active as well.

Cell-bound Notch ligand induces DC maturation

Exposure of DCs to the synthetic Jagged peptide to simulate ligand-induced activation of the Notch signaling pathway may be considered nonphysiological. To determine whether contact between cell-bound Notch ligand and Notch-1 on a DC would result in maturation of DCs as well, we studied two models. Human keratinocytes endogenously express Jagged-1 under physiological conditions, as shown by Western blot analysis (Fig. 3GoA). Immunohistochemical staining showed that Jagged-1 was localized on the cell surface (22). Keratinocytes lack CD40 ligand expression, as determined by FACS analysis (data not shown). Coculture of immature DCs with human keratinocytes resulted in up-regulation of MHC class II, CD80, CD83, and CD86 (Fig. 3GoB). Moreover, these DCs were able to induce T cell proliferation and IFN-{gamma} production by allogeneic T cells (control vs keratinocytes, p < 0.001; Fig. 3Go, C and D). This indicates that cells physiologically expressing Jagged-1 can induce DC maturation in vitro.



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  		FIGURE 3. Physiologically expressed Jagged-1 induces the up-regulation of maturation markers. Keratinocytes, expressing Jagged-1 endogenously, were cocultured with immature DCs for 48 h. Representative experiments are shown of three performed. A, Western blot stained for Jagged-1 of human keratinocytes and vector-transfected and Jagged-1 transfected fibroblasts (MVV and MVJ, respectively). B, Expression levels of activation markers on human DCs 48 h after coculture with keratinocytes as measured by FACS. The black line represents DCs cocultured with keratinocytes, the gray line represents LPS-exposed DCs, and the gray-filled line represents immature DCs. C, MLR using DCs cocultured with LPS or keratinocytes as stimulators and allogeneic PBL as responders. [3H]thymidine incorporation was determined as a measure of T cell proliferation. D, IFN-{gamma} production was determined by ELISA in an MLR using DCs cultured in the presence of LPS or keratinocytes as stimulators and allogeneic PBL as responders.

 
However, this observation per se does not prove that the observed DC maturation is mediated by Jagged-1 expressed on keratinocytes. To further investigate the role of cell-associated Jagged-1, fibroblasts were transfected with human Jagged-1 (MVJ) or empty vector (MVV). Fibroblasts have low endogenous expression of Jagged-1, and transfection of Jagged-1 cDNA significantly increased this expression (Fig. 3GoA).

Jagged-transfected fibroblasts were cocultured with immature DCs, and the expression of activation markers was assessed (Fig. 4GoA). Expression of CD80, CD83, and CD86 on DCs cultured with Jagged-transfected fibroblasts was similar to the expression of these maturation markers on LPS-treated DCs. In contrast, DCs cultured with vector-transfected fibroblasts showed low expression of these markers, similar to control DCs. IL-12 production by DCs cultured with Jagged-transfected fibroblasts was determined 24 h after the start of the coculture and compared with IL-12 production by LPS-treated DCs (Fig. 4GoB). Both LPS- and MVJ-treated DCs produced similar amounts of IL-12 (control vs MVJ, p < 0.001). DCs exposed to control MVV fibroblasts produced significantly less IL-12 (MVV vs MVJ or LPS, p < 0.001). To test the functionality of DCs exposed to Jagged-1-transfected fibroblasts, a mixed lymphocyte reaction was set up (Fig. 4GoC). LPS- or MVJ-cultured DCs induced equal proliferation of allogeneic PBL, as measured in an MLR. In contrast, DCs cocultured in the presence of fibroblasts transfected with empty vector did not increase T cell proliferation (control vs MVJ, p < 0.001). Likewise, IFN-{gamma} production was induced only by DCs exposed to LPS- or Jagged-1-transfected fibroblasts (control vs MVJ, p < 0.01; Fig. 4GoD). Taken together, these data indicate that Notch activation by a soluble ligand or a cell-associated ligand induces phenotypical and functional DC maturation.



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  		FIGURE 4. Cell-bound Jagged-1 induces DC maturation. DCs were cultured on an adherent monolayer of Jagged-1- or vector-transfected fibroblasts, and nonadherent cells were harvested after 48 h. Representative experiments are shown of three performed. A, Expression levels of activation markers on human DCs 48 h after coculture with transfected fibroblasts as measured by FACS. The black line represents DCs cocultured with Jagged-transfected fibroblasts, the light gray line represents LPS-treated DCs, the dark gray line represents DCs cocultured with vector-transfected fibroblasts, and the filled area represents immature control DCs. B, IL-12 (p70) production was measured in DCs cocultured with Jagged-1-transfected (MVJ) or vector-transfected (MVV) cells. C, An MLR where DCs cultured with LPS, Jagged-1 (MVJ), or vector (MVV)-transfected fibroblasts were cocultured with allogeneic PBL. [3H]Thymidine incorporation was determined as a measure of T cell proliferation. D, IFN-{gamma} production was determined by ELISA in an MLR coculture using DCs cultured in the presence of LPS or human fibroblasts transfected with Jagged-1 (MVJ) or vector (MVV) as stimulators and allogeneic PBL as responders.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Notch signaling is involved in multiple lineage decisions in the hemopoietic system. In the lymphoid lineage several developmental choices are thought to be regulated by Notch. The T vs B cell lineage choice (26, 27, 28), the {alpha}{beta} vs {gamma}{delta} T cell choice (29), and the CD4 vs CD8 T cell decision (16, 30, 31) are all thought to be regulated by Notch signaling. In the myeloid lineage Notch signaling has been implicated in the early transition from a granulocyte-monocyte progenitor to differentiated granulocyte, although there is controversy on the exact function of Notch in this cell lineage (32, 33, 34, 35). Evidence obtained in conditional Notch-1 knockout mice suggested that Notch-1 is dispensable during the differentiation from this granulocyte-monocyte progenitor to immature DCs (18). However, in antisense Notch-1 transgenic mice, reduced Notch-1 expression resulted in significantly lower NF-{kappa}B activity (36), which is known to play a major role in DC maturation. This suggested that Notch-1 might be involved in the maturation process of DCs rather than in their development. Here, we show that direct stimulation of the Notch signaling pathway with a soluble or a cell-bound Notch ligand induced DC maturation similar to LPS: Jagged-treated DCs expressed maturation markers were no longer capable of Ag uptake, produced IL-12(p70), and induced T cell proliferation and IFN-{gamma} production. Thus, although Notch-1 is dispensable for DC development, our data provide ample evidence that activation of Notch-1 signaling promotes the maturation of human DCs.

According to a commonly accepted model for Notch signaling, high levels of activated Notch cause transcriptional down-regulation of lineage-specific genes. This results in a delay of cell differentiation and continuation of an uncommitted state until a second differentiation signal determines a different cell fate (37, 38, 39, 40, 41). Our data suggest that Notch activation can also lead to the opposite: transcription of lineage-specific genes and terminal differentiation into mature DCs. It is quite possible that in some mammalian cell lineages a timed activation of one or more Notch receptors controls the timing of cell maturation. Similar observations were made in murine keratinocytes (42) and more recently in human keratinocytes (22). Human keratinocytes express Notch-1, -2, -3, and -4 depending on their maturation state. Treatment with the Jagged peptide triggers terminal differentiation, culminating in cornification. Additionally, Notch-1 signaling is necessary for the differentiation of preadipocytes (43), murine erythroleukemia cells (9), and CD8 single-positive thymocytes (30). The studies mentioned above, like the present one, do not rely on transfected, overexpressed forms of constitutively active Notch receptors, which may have nonphysiological effects. As a separate study has shown that Notch signaling does not affect DC development (18), our data indicate that in DCs, Notch-1 signaling modulates relatively late stages of differentiation, even terminal differentiation, rather than initial cell fate commitment. Therefore, it appears that in several instances, physiologically regulated expression of endogenous Notch receptors controls the timing of cell differentiation and can positively affect differentiation in several cell types, including DCs (44, 45).

Our data indicate that it is possible to induce complete maturation of human DC by activating the Notch signaling pathway. This raises the obvious question of whether DCs are exposed to Notch ligands in the microenvironments where they reside. Knowledge of the tissue expression of Notch ligands is relatively limited. Jagged-1 expression was observed in endothelial cells (46, 47), thymus (48), bone marrow stromal cells (21), and suprabasal keratinocytes. Jagged-2 is expressed in thymus (49). Expression of Delta-3 has been detected in brain (50), and Delta-4 is located in arterial endothelium (51). It is not likely that DCs are induced to mature upon the mere encounter of a Notch ligand expressed on a cell in its immediate environment, such as in bone marrow, skin, thymus, or blood. In skin, keratinocytes constitutively express high levels of Jagged-1. Skin DCs, such as Langerhans cells, that express Notch-3 and -4 (B. Nickoloff, unpublished observations) are not constitutively induced to mature even though our data show that it is possible to induce monocyte-derived DC maturation by keratinocytes or Jagged-1-transfected fibroblasts. Therefore, to prevent constitutive maturation of DCs, Notch signaling must be tightly regulated. It is conceivable that Jagged-Notch signaling is inhibited until physiological conditions require DC maturation. It should be noted that the activity of Notch receptors is modulated by multiple mechanisms that control the maturation of Notch proteins, including the amount of Notch exposed on the membrane through membrane recycling, the activation of transmembrane Notch, and the half-life of intracellular Notch (for a review, see Ref. 52). Potential negative regulators of Notch signaling include the endocytic mediator Numb (53, 54), the Notch-binding protein Notchless (55), and E3 ubiquitin-ligases such as SEL-10 (56, 57). The affinity of Notch receptors for their ligands is further modulated by the Fringe family glycosyltransferases (58).

A more detailed analysis of the downstream targets of the LPS signaling pathway needs to be performed to determine whether and how signaling triggered by TLR receptors and/or CD14 cross-talks with Notch signaling. Based on our data and previous reports (36, 59), it is likely that NF-{kappa}B cross-talks with the Notch signaling pathway. NF-{kappa}B plays a major role in signaling cascades that lead to maturation of DCs. Most maturation signals, such as CD40 (60), TNF-{alpha} (6), and TLR (6), involve NF-{kappa}B. Recent data indicate that Notch-1 is required for the expression of several NF-{kappa}B subunits in murine hemopoietic precursors (36). Moreover, we have observed that Jagged-1 peptide treatment of human keratinocytes leads to rapid activation of NF-{kappa}B (22). Additionally, NF-{kappa}B has been proposed to trans-activate Jagged-1, one of the Notch ligands (61). Taken together, these data suggest that Notch-1 may enhance NF-{kappa}B-dependent signals and, in turn, NF-{kappa}B may amplify the effect by up-regulating Notch ligand Jagged-1. The latter may act cell-autonomously or trans-cellularly to further activate Notch-1.

The present observations have potentially important medical implications. Activation of Notch-1 may be a viable strategy for in vivo and ex vivo immunomodulation in applications such as DC-based tumor vaccines or vaccines against infectious agents or toxins.


   Acknowledgments
 
We thank Dr. Spyros Artavanis-Tsakonas for the generous gift of plasmid containing human Jagged-1 and Dr. Stephen Meredith for synthesis of the Jagged and Scrambled peptides. We are grateful to Erica J. Simel for her assistance with some DC maturation experiments.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AR40065 and PO1-CA-59327 (to B.J.N.), RO1CA84065-2 (to L.M.), and RO1CA/AI78399 (to W.M.K.). M.P.V. is a fellow of the Cancer Research Institute. Back

2 Address correspondence and reprint requests to Dr. W. Martin Kast, Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153. E-mail address: mkast{at}lumc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; NIC, intracellular Notch; TLR, Toll-like receptor. Back

Received for publication May 30, 2002. Accepted for publication August 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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