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The Journal of Immunology, 2000, 165: 3647-3655.
Copyright © 2000 by The American Association of Immunologists

Bacterial Lipopolysaccharide, TNF-{alpha}, and Calcium Ionophore Under Serum-Free Conditions Promote Rapid Dendritic Cell-Like Differentiation in CD14+ Monocytes Through Distinct Pathways That Activate NF-{kappa}B

Lyudmila A. Lyakh1,*, Gary K. Koski1,*, William Telford{dagger}, Ronald E. Gress{dagger}, Peter A. Cohen{ddagger} and Nancy R. Rice2,*

* Division of Basic Sciences, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702; {dagger} Medicine Branch, National Cancer Institute, Bethesda, MD 20814; and {ddagger} The Center for Surgery Research, Cleveland Clinic Foundation, Cleveland, OH 44195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-{alpha}, or calcium ionophore (CI). Within 48–96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-{alpha} production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-{kappa}B family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-{kappa}B. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myeloid dendritic cells (DC)3 promote the activation of T lymphocytes for both primary and recall responses to Ag. A current paradigm (1, 2) holds that DC arise from proliferating precursors found in the bone marrow. These cells enter the blood as a population(s) of nonproliferating DC precursors, which almost certainly includes CD14+ monocytes. These cells may then permeate peripheral tissues, including the skin, where they serve a sentinel function awaiting signals that indicate local infection or inflammation (3). These signals may include bacterial cell wall components such as LPS or proinflammatory cytokines such as TNF-{alpha} (4). Upon receiving such signals, DC enter a terminal differentiation/activation program, during which they up-regulate activation, MHC, and T cell costimulatory molecules and quickly leave peripheral tissues, presumably taking with them an antigenic sample of their local environment. DC then enter the afferent lymphatics and migrate to the T cell-dependent areas of the draining lymph nodes. Here they form associations with Ag-specific T lymphocytes, thereby initiating T cell-dependent immune responses. Transcription factors of the Rel/NF-{kappa}B family, including p50, p52, and RelB, are expressed by and play an important role in the biology of DCs (5).

DCs derived from CD14+ monocytes are often obtained in vitro first by culturing these cells in FCS- or human serum-containing medium supplemented with a combination of GM-CSF and IL-4 (6). Monocytes cultured under these conditions for 1 wk or more down-regulate surface CD14 and enter a developmental state comparable to that of immature, tissue-resident DC. To promote the attainment of full DC maturity, additional agents, such as bacterial LPS, TNF-{alpha}, and monocyte-conditioned medium, have been used successfully (7, 8, 9). We have recently demonstrated that Ca2+-mobilizing agents can also induce the differentiation of DC from CD14+ monocytes (10). This Ca2+-dependent differentiation process is extremely rapid (1–2 days) and is sensitive to calcineurin antagonists, including cyclosporin A (CsA) (11).

Our primary interest is elucidation of the signaling pathways involved in the differentiation of DC from CD14+ precursors. Such studies would be facilitated if the differentiation period of DC could be cut from 1–2 wk to only a few days. Another inducement for the discovery of rapid DC culture methods is the potential for use of DC in immunotherapy. Cell-based therapies aimed at treating malignancies or infections demand that time in culture be kept to a practical minimum. In fact, ideal culture systems would be not only rapid, but free of animal or human serum, because issues of safety and consistency are paramount. For these reasons, we have developed a compositionally simplified serum-free culture system that can rapidly (4 days or less) yield mature DC from human CD14+ monocytes using a variety of agents to drive differentiation.

In the studies presented here we show that under serum-free conditions, LPS, TNF-{alpha}, and calcium ionophore (CI) A23187 are each capable of inducing rapid DC differentiation in human monocytes. Most interestingly, we show that CI treatment leads to the induction of NF-{kappa}B proteins, particularly RelB, in myeloid cells. Despite the Ca2+ dependence and CsA sensitivity of CI-induced differentiation, we found no evidence of NF-AT involvement in this pathway. Although LPS, TNF-{alpha}, and A23187 all induced DC differentiation, there must be significant differences in the signaling pathways used, because 1) TNF-{alpha} production is required for LPS-induced, but not for A23187-induced, differentiation; and 2) CsA blocks differentiation induced by A23187, but not that induced by LPS. However, in all cases expression of the DC activation marker CD83 was associated with enhanced levels of nuclear NF-{kappa}B. Thus, despite upstream differences, the signaling pathways that induce DC differentiation each activate NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
mAb and FACS analysis

The methods employed and the mAb specific for human CD80, CD86, CD14, CD83, CD40, and HLA-DR as well as isotype-matched control mAbs were identical with those used in previously published studies (11). A gate (R1) was employed in all FACS analysis (except FACS separation experiments) to include all viable cells based on lack of propidium iodide staining. The instrumentation employed in flow cytometry studies was a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) running CellQuest analysis software. For separation of CD14+CD83- from CD14-CD83+ cells, LPS-treated monocytes were double stained with FITC-anti-CD14 and PE-anti-CD83, washed, and resuspended in PBS with 2% FCS at 5 x 106 cells/ml. Cells were sorted using a Becton Dickinson FACSvantage SE high speed fluorescence-activated cell sorter. The resulting populations were confirmed by back-analysis to be >98% pure.

Culture of human peripheral blood monocytes, T cells, and cell lines

Human CD14+ peripheral blood monocytes (92–95% purity) were obtained from 16 healthy donors by leukapheresis and elutriation according to National Institutes of Health guidelines for human subjects, and either cultured immediately or cryopreserved as described previously (10). Lymphocyte-rich fractions were also collected, and T cells for allosensitization studies were purified using T cell isolation columns (R&D Systems, Minneapolis, MN). Monocytes were plated at a density of 2.5 x 106 cells/well in 24-well tissue culture plates (Costar, Corning, NY) in 2 ml/well macrophage serum-free medium (M{phi}-SFM; Life Technologies, Gaithersburg, MD) supplemented with 50 ng/ml recombinant human GM-CSF (Immunex, Seattle, WA), as described previously (11). Cells cultured overnight were induced to differentiate by addition of varying doses of LPS derived from Escherichia coli strain O26:B6 (Sigma, St. Louis, MO), 100–200 U/ml TNF-{alpha} (PeproTech, Rocky Hill, NJ), or 188–225 ng/ml CI A23187 (Sigma). Because DC differentiation was achieved more rapidly with CI than with either LPS or TNF-{alpha} (24 h vs up to 72 h), in experiments aimed at immunophenotypical characterization, cell morphology, and function, CI addition was delayed for 2 days so that at the end of a 96-h total culture period all treatment groups would be developmentally synchronized. Signaling pathway inhibitors included CsA (Sandoz, Basel, Switzerland), N-acetyl cysteine (NAC; Sigma), and a TNF-{alpha}-neutralizing mAb 5N (12). The human promyelocytic leukemia line, HL-60 (CCL 240), was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI medium with 10% FCS (HyClone, Logan, UT) as described previously (13).

Measurement of TNF-{alpha} in monocyte culture supernatants

Monocytes cultured overnight in M{phi}-SFM supplemented with GM-CSF were treated with 50 ng/ml LPS or 188 ng/ml A23187. After 24 h, culture supernatants were removed and assayed by ELISA (R&D Systems) for the presence of TNF-{alpha} by the Lymphokine Testing Laboratory, Frederick Cancer Research and Development Center, National Cancer Institute (Frederick, MD). The limit of detection of this assay is 15 pg/ml TNF-{alpha}.

Antisera

Rabbit antisera that recognize human cRel (serum 265), human p50 (serum 1141), and NF-AT (serum 796) have been described previously (14, 15). RelB antisera were raised against the following peptides: 1319, NH2-CREAAFGGGLLSPGPEAT, located at the C terminus of human RelB; and 1393, NH2-LRSGPASGPSVPTGRC, located at the N terminus of human RelB (16). The cysteine residues were added to facilitate coupling of the peptides to hemocyanin.

Oligonucleotides

For the EMSA we used oligonucleotides containing binding sites for NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega, Madison, WI), octamer-1 (5'-TGT CGA ATG CAA ATC ACT AGA A-3'; Promega), and NF-AT (5'-ATA AAA TTT TCC AAT GTA AA-3'). The double-stranded oligonucleotides were labeled to high specific activity with [{gamma}-32P]ATP (6000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) and T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN).

Preparation of cellular extracts and EMSA

Cells were rinsed and resuspended in ice-cold hypotonic buffer (25 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 5 mM KCl, and protease inhibitor mixture (Roche Molecular Biochemicals)) at a concentration of 1–2 x 107 cells/ml, incubated on ice for 15 min, and lysed with an equal volume of hypotonic buffer containing 0.3% Nonidet P-40, additional protease inhibitor {alpha}2-macroglobulin (2 U/ml; Roche Molecular Biochemicals), and phosphatase inhibitors (1 mM sodium vanadate and 1 µM okadaic acid). The lysate was centrifuged at 500 x g for 5 min; the supernatant constitutes the cytoplasmic fraction. The pelleted nuclei were extracted with 30–50 µl of a solution containing 10 mM Tris-HCl (pH 7), 220 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 0.05% Nonidet P-40, and inhibitors at 4°C for 20 min with agitation. The extract was centrifuged at 15,000 x g for 15 min at 4°C; the supernatant constitutes the nuclear extract.

For EMSA, the binding reaction mixture was 10 mM HEPES (pH 7.5), 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 6% glycerol, 0.5 µg of polydeoxyinosinic-deoxycytidylic acid (poly(dI-dC)), 0.5 µg of sonicated double-stranded salmon sperm DNA, 32P-labeled oligonucleotide (1 x 105 to 3 x 105 cpm), and nuclear extract (2–3 µg of protein) in a total volume of 20 µl. The mixture was incubated at room temperature for 15 min. For supershift analysis, the reaction mixture minus 32P-labeled DNA was preincubated for 15 min on ice with 1 µl of a 1:3 dilution of antiserum. The 32P-labeled oligonucleotide was then added, and the product was analyzed on 6% DNA retardation gels (Invitrogen/NOVEX, Carlsbad, CA).

Immunoprecipitation and immunoblotting

Extracts were incubated overnight with antiserum and protein A-Sepharose (Amersham Pharmacia Biotech) after dilution with TNT buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 1% Triton X-100). Washed precipitates were resolved by 10% Tricine SDS-PAGE (NOVEX) and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Immunoreactive proteins were revealed with an enhanced chemiluminescent system (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Under serum-free conditions LPS, TNF-{alpha}, and A23187 each rapidly induce mature DC immunophenotype in a majority of CD14+ monocytes

We previously showed that A23187 induced rapid differentiation of DC from monocytes under serum-free conditions (11) and began these studies by testing the differentiating effects of LPS and TNF-{alpha} under identical conditions. As expected, uncultured, elutriated monocytes almost uniformly expressed surface CD14, CD86, and HLA-DR, but were largely low/negative for the expression of CD83, CD80, and CD40 (Fig. 1Go). To maintain the viability of these cells in M{phi}-SFM, the addition of GM-CSF proved necessary. Culture for 96 h under these conditions usually led to a loss of surface CD86 and to a slight to moderate down-regulation of CD14 expression. Moderate up-regulation of CD80 and CD40 was also observed, but there was little effect on HLA-DR expression, and no effect on the expression of the DC activation marker CD83. Markedly different results were obtained when Escherichia coli O26:B6 LPS at 50 ng/ml was included for the final 72 h of culture (Fig. 1Go). Between 50 and 80% of the cells from all donors lost surface CD14, and these cells showed marked up-regulation of CD83. Greatly enhanced expression of CD80, CD86, CD40, and HLA-DR was also observed compared with that of controls treated with GM-CSF only. Dose-response studies indicated that monocytes cultured in M{phi}-SFM were very sensitive to LPS (data not shown). Plateau responses were seen in the dose range of 10–100 ng/ml, although a response was still evident at concentrations as low as 0.1 ng/ml LPS. In two-color analysis, it was clear that the subpopulation of cells that became negative for the monocyte/macrophage marker CD14 were always the highest expressers of CD80, HLA-DR, and the DC activation marker CD83, consistent with this population’s identity as DC (not shown).



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  		FIGURE 1. Bacterial LPS, TNF-{alpha}, and A23187 induce the DC immunophenotype in CD14+ monocytes. Monocytes were cultured overnight in M{phi}-SFM supplemented with 50 ng/ml GM-CSF. The next day, 50 ng/ml LPS or TNF-{alpha} (200 U/ml) was added. Calcium ionophore A23187 (188 ng/ml) was added to respective wells 48 h later. After a total 96-h culture period, cells were harvested and analyzed by FACS. {square}, Isotype-matched control Ab staining; {blacksquare}, marker-specific Ab. Results are representative of six experiments with different donors.

 
TNF-{alpha} likewise displayed dramatic effects (Fig. 1Go). In most donors, TNF-{alpha} caused a loss of CD14 expression that was much more uniform than that induced by LPS. A majority of cells expressed CD83, and marked up-regulation of CD80, CD86, CD40, and HLA-DR was also observed. The effects of A23187 were the most rapid and complete. After only 24-h treatment, there was near total down-regulation of CD14 and uniform up-regulation of CD83 expression. As with LPS and TNF-{alpha}, A23187 also enhanced the expression of CD80, CD86, and HLA-DR, but the CD40 increase was sometimes slightly less than that seen with the other treatments. Results from five separate experiments showed that differentiation treatments did not cause an unacceptable loss of viability over the 96-h culture period. Relative to undifferentiated cells, viabilities were 99 ± 3% for LPS treatment, 79 ± 7% for TNF-{alpha} treatment, and 74 ± 4% for CI treatment. Thus, LPS, TNF-{alpha}, and A23187 are each capable of inducing rapid conversion of CD14+ peripheral blood monocytes into cells with a DC-like immunophenotype under serum-free conditions.

Human AB serum has an inhibitory effect on DC differentiation

Most methods using cytokines for deriving mature DC from CD14+ monocytic precursors use serum-containing medium and require 1–2 wk of culture. We tested whether the more rapid kinetics we observed were due to some positive factor(s) supplied by the M{phi}-SFM or to an inhibitory factor(s) found in serum. DC differentiation was induced by TNF-{alpha} or LPS in elutriated monocytes as described above, except that some groups were cultured in M{phi}-SFM supplemented with 5% human AB serum. The starting (uncultured) population was 92% positive for surface CD14 and low/negative for CD83 (Fig. 2Go, population defined by R2), and only about 1% of the cells had a CD14-CD83+ mature DC phenotype (defined by R3). Treatment with either LPS or TNF-{alpha} in M{phi}-SFM resulted in 71% of the cells converting to the CD14-CD83+ phenotype. However, inclusion of 5% serum resulted in decrease of the CD14-CD83+ cells from 71 to 7% (LPS treatment) and from 71 to 1% (TNF-{alpha} treatment). Viability remained very high in all cultures, ranging from 79 to 91% for the LPS- and TNF-{alpha}-treated samples. Similar inhibitory effects were observed in three separate lots of human serum from two different suppliers. Suppression of rapid differentiation was also seen when FCS was used instead of human serum (data not shown). These results suggest that under our experimental conditions the absence of serum facilitates rapid DC differentiation induced by LPS or TNF-{alpha}.



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  		FIGURE 2. Human serum inhibits DC differentiation. Elutriated human monocytes were cultured for 1 day in M{phi}-SFM supplemented with GM-CSF with or without 5% human AB serum as indicated. LPS (50 ng/ml) or TNF-{alpha} (200 U/ml) was then added, and cultures were harvested for FACS analysis 72 h later. Cells were stained with FITC- or PE-labeled Abs specific for CD14 (vertical axis) or CD83 (horizontal axis). R2, Cells that are CD14+, low/negative for CD83; R3, cells that are CD14 low/negative, CD83+. Results are representative of three experiments.

 
LPS-, TNF-{alpha}-, and A23187-treated monocytes acquire DC morphology

To examine the morphological changes induced by various treatments, CD14+ peripheral blood monocytes were cultured in M{phi}-SFM plus GM-CSF with or without LPS, TNF-{alpha}, or A23187 as described above. At the end of the 96-h culture period, cells from all treatment groups were harvested, and cytospin preparations were made onto glass slides and Wright stained. Dendritic morphology, including the characteristic cellular processes, was virtually absent on cells cultured in M{phi}-SFM supplemented only with GM-CSF (Fig. 3GoA). In contrast, such processes were clearly apparent on a majority of cells treated with LPS (Fig. 3GoB), TNF-{alpha} (Fig. 3GoC), and A23187 (Fig. 3GoD).



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  		FIGURE 3. Human CD14+ monocytes exhibit DC morphology after treatment with LPS, TNF-{alpha}, or A23187. Monocytes were treated as described in Fig. 1Go. After a total 96-h culture period, cells were harvested, washed, and resuspended in HBSS. Cytospin preparations were made onto glass slides, Wright stained, and subjected to photomicrography. A, GM-CSF alone; B, GM-CSF plus LPS; C, GM-CSF plus TNF-{alpha}; D, GM-CSF plus A23187. Results are representative of three separate experiments.

 
LPS-, TNF-{alpha}-, and A23187-treated monocytes acquire enhanced allostimulatory capacity

We next tested uncultured and cultured cells for their capacity to stimulate T lymphocytes in the allogenic MLR. Uncultured cryopreserved monocytes (Fig. 4Go) demonstrated a relatively poor capacity to stimulate T cells, with appreciable proliferation only induced at APC:T cell ratios of 1:25 or less. In contrast, cells cultured in M{phi}-SFM with GM-CSF alone exhibited markedly enhanced allosensitizing capacity. Although these cells lack many features characteristic of DCs, this enhanced capacity to allosensitize T cells may rest on the demonstrated induction of costimulatory molecules such as CD80 and CD40 (Fig. 1Go). Nevertheless, the most efficient cells at stimulating T cell proliferation in the allogenic MLR were those cells with activated DC characteristics: LPS-, TNF-{alpha}-, and A23187-treated monocytes. As expected, combinations of autologous T cells and APC resulted in almost no [3H]thymidine incorporation. These results show that in the absence of serum, human monocytes can acquire enhanced APC function consistent with DC.



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  		FIGURE 4. Bacterial LPS, TNF-{alpha}, and A23187 induce enhanced allosensitizing capacity in human CD14+ monocytes. Graded numbers of gamma-irradiated (30 Gy) untreated monocytes ( and ) or monocytes cultured in M{phi}-SFM supplemented with GM-CSF ({circ} and •), GM-CSF plus LPS ({diamond} and {diamondsuit}), GM-CSF plus TNF-{alpha} ({triangleup} and {blacktriangleup}), or GM-CSF plus A23187 ({square} and {blacksquare}) were added to 96-well tissue culture plates with purified allogenic (open symbols) or autologous (filled symbols) T lymphocytes (2 x 105/well). Cells were cocultured for 96 h, then pulsed with 1 µCi of [3H]TdR for an additional 18 h, and [3H]TdR incorporation in harvested cells was assessed by liquid scintillation spectrometry. Results are representative of three separate experiments with different donors.

 
TNF-{alpha} neutralizing Ab inhibits acquisition of DC immunophenotype in monocytes treated with TNF-{alpha} or LPS, but not A23187

Having established that activated DC characteristics can be rapidly induced in normal human monocytes under serum-free conditions, we turned our attention to the signaling pathways involved in this process. Because TNF-{alpha} secretion is a characteristic response of monocytes to LPS (17, 18), and because TNF-{alpha} has a differentiating effect on monocyte-derived DC (7, 9), we asked whether LPS induces DC differentiation in monocytes wholly or in part through the induced secretion of TNF-{alpha}. We found that monocytes cultured in M{phi}-SFM with GM-CSF and LPS produced high levels of TNF-{alpha} (typically on the order of 30–40 ng/ml in 24 h). A23187-treated cells also produced TNF-{alpha}, but at a level almost 200-fold lower (200 pg/ml) than that induced by LPS. We then asked whether DC differentiation could be inhibited by TNF-{alpha}-neutralizing Ab. We found that, as expected, the Ab completely inhibited the acquisition of DC immunophenotype in cells treated with TNF-{alpha} (Fig. 5Go). Up-regulation of CD83, CD80, and HLA-DR was abrogated, as was down-regulation of CD14. When cells were treated with LPS, the Ab also had a dampening effect. Although the inhibition was not as complete as that with TNF-{alpha}, a nonetheless marked reversal of CD14 loss as well as suppressed up-regulation of CD83, CD80, and HLA-DR were evident, suggesting an important contribution from TNF-{alpha} in the LPS-mediated conversion of monocytes to activated DC under the conditions tested. In contrast, with A23187-treated cells the TNF-{alpha}-neutralizing Ab had little or no effect on CD14 loss or on CD83, CD80, and HLA-DR up-regulation. This suggests that TNF-{alpha} is not required for A23187-induced DC differentiation, and that A23187 acts either downstream of TNF receptor 1 (TNFR1) in the TNF signaling pathway or through a different pathway altogether.



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  		FIGURE 5. Neutralizing TNF-{alpha} Ab abrogates the DC-differentiating effect of TNF-{alpha} and LPS, but not that of A23187. Monocytes were cultured overnight in M{phi}-SFM supplemented with GM-CSF. Immediately before the addition of LPS (1 ng/ml), TNF-{alpha} (200 U/ml), or A23187 (188 ng/ml), 5 µg/ml of 5N, a TNF-{alpha}-neutralizing Ab, was added. After a total 96-h culture period, cells were stained with FITC- or PE-labeled mouse mAbs specific for CD14 (vertical axis in dot plots), CD83 (horizontal axis), CD80, or HLA-DR and analyzed by FACS. {square}, Isotype-matched control Ab staining; {blacksquare}, marker-specific Ab. Results are representative of three experiments.

 
Treatments that promote DC differentiation from monocytes activate nuclear NF-{kappa}B through pathways differentially sensitive to CsA

Previous studies have implicated NF-{kappa}B transcription factors in the process of DC differentiation (19, 20, 21). To test whether NF-{kappa}B is activated under serum-free conditions, monocytes were treated with LPS or A23187 for 24 h, and nuclear proteins were analyzed by EMSA using an NF-{kappa}B consensus oligonucleotide. Extracts from cells cultured in M{phi}-SFM plus GM-CSF had a low level of nuclear DNA-binding activity (Fig. 6GoA, lane 1). In contrast, treatment of the cells with LPS (lane 2) or A23187 (lane 5) for 24 h resulted in a high level of nuclear NF-{kappa}B DNA-binding activity, with LPS clearly the more potent inducer of NF-{kappa}B. TNF-{alpha} treatment also resulted in elevated NF-{kappa}B DNA binding (data not shown).



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  		FIGURE 6. Differential sensitivity of NF-{kappa}B induction to CsA. A, Human monocytes were cultured in M{phi}-SFM supplemented with GM-CSF for 24 h. Cells were then treated with LPS (50 ng/ml) or A23187 (188 ng/ml) with or without pretreatment with NAC (40 mM, 1.5-h pretreatment) or CsA (500 ng/ml, 18-h pretreatment). HL-60 cells were treated with A23187 (263 ng/ml) with or without pretreatment with CsA (500 ng/ml, 45-min pretreatment). After LPS or A23187 treatment for 24 h, nuclear extracts were analyzed by EMSA using the 32P-labeled NF-{kappa}B consensus binding site. B, The same monocyte nuclear extracts (25 µg of protein) as those used in A were immunoprecipitated with anti-p50, separated by SDS-PAGE, transferred to nitrocellulose, and probed with a combination of anti-RelB serum 1319 and anti c-Rel serum 265. HL-60 nuclear extracts (30 µg of protein) were precipitated with anti-Rel B serum 1319. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti RelB serum 1319. C, Levels of surface CD83 after 24 h of treatment are displayed in bar graph form as an expression index, which is defined as the geometric mean fluorescence of cells stained with PE-labeled anti-CD83 Ab divided by the geometric mean fluorescence of cells stained with an isotype-matched control Ab. An expression index of 1 indicates that fluorescent intensity is equal to the negative control value. Experimental results representative of four experiments are shown.

 
It has been demonstrated previously that treatment of the HL-60 promyelocytic cell line with Ca2+ mobilizers, but not other agents, led to the acquisition of immunophenotypical, morphological, and functional properties similar to those of DC (13). Untreated HL-60 cells had little or no constitutive nuclear NF-{kappa}B DNA-binding activity (Fig. 6GoA, lane 7). However, after treatment of cells with A23187 for 24 h, a high level of DNA-binding activity was detected in nuclear extracts (lane 9). Thus, all treatments (LPS, TNF-{alpha}, A23187) that led to acquisition of DC phenotype in either monocytes or HL-60 cells also resulted in induction of nuclear NF-{kappa}B DNA-binding activity.

To gain insight into the mechanism of activation of NF-{kappa}B by LPS and A23187, we tested the effect of CsA, which inhibits the calcium/calmodulin-dependent phosphatase calcineurin. We found that the pretreatment of monocytes with CsA blocked A23187-induced NF-{kappa}B DNA binding activity (Fig. 6GoA), nuclear expression of cRel and RelB proteins (Fig. 6GoB), and surface expression of the DC activation marker, CD83 (Fig. 6GoC, compare lanes 5 and 6 in all panels). Similar results were obtained with HL-60 cells (compare lanes 9 and 8 in all panels). However, CsA had no detectable inhibitory effect on LPS-induced differentiation (Fig. 6Go, compare lanes 2 and 4). In contrast, induction by LPS of nuclear NF-{kappa}B and surface expression of CD83 were efficiently blocked by NAC, a potent inhibitor of NF-{kappa}B activation (Fig. 6Go, lanes 2 and 3). (The effect of NAC on either A23187-treated monocytes or HL-60 cells could not be assessed due to severe combined toxicity of NAC and A23187.) Thus, in all cases the expression of CD83, a marker for DC differentiation, was associated with the expression of nuclear NF-{kappa}B. These data are consistent with the hypothesis that NF-{kappa}B activity is closely associated with the differentiation process. Furthermore, the differential sensitivity of LPS- and A23187-treated cells to CsA offers strong evidence that there are differences in the upstream signaling pathways activated by these inducers despite common activation of NF-{kappa}B proteins. Also of considerable interest is our observation that RelB (Fig. 6GoB), which is known to be associated with DC differentiation, is induced not only by LPS and TNF-{alpha} (not shown) but also by A23187, a Ca2+-mobilizing agent.

The purified CD83+/CD14- population from LPS-treated monocytes expresses high levels of nuclear NF-{kappa}B, including RelB

As shown above, LPS treatment of CD14+ human monocytes leads to the loss of CD14 expression and the concomitant expression of the DC activation marker CD83 in a sizable fraction, but not all, of the cells. To determine whether nuclear NF-{kappa}B is associated preferentially with the DC-like population, LPS-treated human monocytes were separated by FACS into CD83+/CD14- and CD83-/CD14+ subpopulations (Fig. 7GoA), and nuclear extracts were analyzed by EMSA using the NF-{kappa}B consensus oligonucleotide. Although both subpopulations of these cells had demonstrable nuclear NF-{kappa}B DNA-binding activity (Fig. 7GoB, lanes 1 and 3), the CD83+/CD14- fraction showed by far the higher level (lane 3). The specificity of DNA binding was confirmed by supershift analysis with anti-p50 serum (lanes 2 and 4). Nuclear extracts from the two subpopulations were also analyzed by Western blot (Fig. 7GoD). As expected, nuclear RelB was predominantly detected in the CD83+/CD14- fraction (compare lanes 1 and 2). DNA binding of the ubiquitously expressed transcription factor OCT-1 was identical in the two subpopulations, demonstrating the integrity of the nuclear extracts (Fig. 7GoC). These studies show that the cells responding to LPS under serum-free conditions by acquiring a DC characteristics also, as a group, displayed a higher level of NF-{kappa}B activation and nuclear RelB expression compared with cells retaining the monocyte/macrophage immunophenotype.



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  		FIGURE 7. The purified CD83+/CD14- fraction from LPS-treated human monocytes expresses higher levels of nuclear NF-{kappa}B. Human monocytes cultured in M{phi}-SFM supplemented with GM-CSF for 24 h were treated with LPS (50 ng/ml) for an additional 48 h. Results are representative of three experiments. A, FACS analysis of treated cells, stained with FITC-anti-CD14 and PE-anti-CD83. Subpopulations of CD83-/CD14+ (G1) and CD83+/CD14- (G2) were collected (~2 x 106 cells each). B, Nuclear extracts (3 µg of protein) from both subpopulations were analyzed by EMSA using a 32P-oligonucleotide containing the NF-{kappa}B binding site. Supershift analysis was performed with anti-p50 serum (lanes 2 and 4). C, The same nuclear extracts (3 µg of protein) were analyzed by EMSA using an OCT-1 consensus probe. D, Nuclear extracts (5 µg of protein) were immunoprecipitated with anti-RelB serum 1319, fractionated by SDS-PAGE, blotted, and probed with serum 1319.

 
Size heterogeneity of human RelB

As shown in Figs. 6GoB and 7D, anti-RelB serum 1319 detected two proteins of very similar size in both human monocytes and HL-60 cells. Inspection of the human RelB cDNA sequence (16) revealed that the 5'-most in-frame ATG lies in a suboptimal context for translation initiation (22), while the second in-frame ATG is located in a better context. If translation can begin at either ATG, two forms of the protein are predicted, differing in size by 17 aa. Antiserum 1319, which was raised against a peptide at the C terminus of the protein, would detect both forms. To test the prediction, we used anti-RelB serum 1393, raised against a 15-residue peptide starting at the first ATG. This serum would be expected to detect only the longer of the two proteins.

We tested antisera 1319 and 1393 on lysates of cells transiently transfected with human RelB as well as on lysates of LPS-treated human monocytes. In both cases immunoprecipitation with serum 1319 resulted in two bands, while precipitation with serum 1393 yielded only the upper band (Fig. 8Go, lanes 2, 3, 5, and 6). Competition with cognate peptide demonstrated the specificity of the recognition (lanes 1 and 4). These results are completely consistent with the prediction of two possible initiation codons in human RelB mRNA, and they indicate that differentiating human monocytes express at least two forms of the RelB protein.



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  		FIGURE 8. Heterogeneity in size of the human RelB protein. HEK 293 cells were transfected with human RelB cDNA, and whole cell extracts were immunoprecipitated with anti-RelB serum 1319 (lanes 1 and 2) or anti-RelB serum 1393 (lanes 3 and 4). Cognate peptide was included for lanes 1 and 4. Cytosolic extracts from human monocytes treated for 24 h with LPS (50 ng/ml) were immunoprecipitated with serum 1319 (lane 5) or serum 1393 (lane 6). Washed precipitates were fractionated by SDS-PAGE, and the immunoblot was developed with serum 1319. Results are representative of three experiments.

 
NF-AT is apparently not involved in DC differentiation from monocytes

The transcription factor NF-AT has been found in variety of cell types, including T cells, B cells, mast cells, and NK cells. NF-AT is activated in these cells by agents that increase intracellular Ca2+ flux. The increased Ca2+ activates the phosphatase calcineurin, which results in dephosphorylation and nuclear translocation of pre-existing cytoplasmic NF-AT and up-regulation of NF-AT synthesis (23, 24). Because CI promotes DC differentiation in monocytes through a calcineurin antagonist-sensitive pathway, we assessed the possible role of NF-AT in this process.

To minimize contamination with lymphocytes, elutriated monocytes were further purified by positive selection for CD14. CD14+ cells were cultured with or without A23187 for 5 or 24 h, and nuclear and cytoplasmic extracts were tested by EMSA for the ability to bind an NF-AT probe. No NF-AT DNA-binding activity was observed in either nuclear or cytoplasmic extracts from these cells (Fig. 9GoA, lanes 1–6). As a positive control, we tested whole cell extracts from human lymphocytes, which, as expected, contained a high level of NF-AT DNA-binding activity (lane 7). A pan-NF-AT antiserum blocked this binding completely, demonstrating the specificity of the interaction (lane 8). Integrity of the monocyte nuclear extracts was confirmed by EMSA with an OCT-1 binding site (Fig. 9GoB). Finally, we were unable to detect NF-AT protein in the treated monocytes by immunoblot analysis (data not shown). Thus, we found no evidence of NF-AT involvement in A23187-induced DC differentiation of monocytes.



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  		FIGURE 9. There is little or no NF-AT in A23187-treated monocytes. Elutriated monocytes were further purified by positive selection with CD14 MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instruction. Cells were then cultured in M{phi}-SFM and GM-CSF, with (lanes 2 and 4–6) or without (lanes 1 and 3) A23187 for 5 or 24 h. Results are representative of three experiments. A, Nuclear (3 µg) and cytoplasmic (10 µg) extracts were tested by EMSA with the NF-AT DNA binding site from the murine IL-4 promoter (a site that can bind NF-AT even in the absence of the AP-1 transcription factor). Whole cell extract (10 µg) from untreated human PBLs was also tested with (lane 8) or without (lane 7) an antiserum (796) that recognizes all NF-AT family proteins. B, The same extracts were tested for the ability to bind an OCT-1 probe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
DCs were originally thought to arise exclusively from precommitted precursor cells. Considerable evidence now exists that CD14+ monocytes, once thought to be chiefly precursors of macrophages, can also give rise to DC. Important recent advances in our understanding of how human monocytes can acquire DC characteristics occurred with the development of an in vitro trans-endothelial cell trafficking model (25), and an in vivo murine model linked to the phagocytosis of intracutaneously injected fluorescent microspheres (26). Both studies showed that a sizable proportion of monocytes could be induced to adopt DC characteristics over the course of about 2–4 days. Although the mechanisms remain unknown, the power of these models is derived from the linkage of plausible physiologic behavior of monocytes to the relatively rapid acquisition of DC characteristics.

Our demonstration of similar rapid differentiation induced by agents known to signal infection or inflammation lends additional credibility to the idea that monocytes indeed contribute to the generation of mature, activated DC in vivo. Our studies clearly show that under serum-free conditions both TNF-{alpha} and bacterial LPS can promote rapid DC differentiation in a majority of CD14+ human peripheral blood monocytes. The treated cells exhibited immunophenotypical, morphological, and functional characteristics of DC while maintaining good viability. Thus, treatment under serum-free conditions may offer a useful alternative to the more typical regimen involving longer culture in medium with serum and IL-4. Given the current interest in culturing DC for therapeutic purposes, both the speed of the differentiation process and the elimination of serum may offer considerable advantages. In fact, when we supplemented our M{phi}-SFM base medium with 5% human AB serum, rapid DC differentiation in response to LPS and TNF-{alpha} was not observed. This suggests that human serum may actually contain components that retard the generation of DC from monocytes under the conditions tested.

Differentiation of DC under serum-free conditions can be driven not only by LPS and cytokines, but also, as we showed previously (11), by CI. In the present studies we demonstrated that the CI-induced differentiation process differs in several respects from that induced by LPS. First, LPS, but not CI, induced high levels of TNF-{alpha} secretion. Second, TNF-{alpha} played a major role in LPS-induced differentiation, for TNF-{alpha}-neutralizing Ab significantly inhibited this process. In contrast, neutralizing Ab had no effect on CI-induced differentiation. This suggests that either CI acts downstream of TNFR1 in the TNF signaling pathway, or it activates a separate pathway altogether. Third, CI-induced differentiation was strongly blocked by CsA, but this drug had no effect on LPS-induced differentiation. This experiment offers evidence that the phosphatase calcineurin plays a vital role in the former pathway, but is not required in the latter.

Regarding the role of TNF-{alpha} in LPS-induced differentiation of DC, our results suggest two possibilities. The first is that TNF-{alpha} is primarily responsible for the differentiation process, and beyond stimulating TNF-{alpha} production, LPS has no additional role to play. This possibility is consistent with the fact that TNF-{alpha} by itself induces DC differentiation, and TNF-{alpha}-neutralizing Ab effectively inhibits LPS-induced differentiation. Alternatively, LPS may activate discrete pathways that modify DC differentiation, resulting in DC with functional capacities different from those of cells differentiated with TNF-{alpha} alone. This possibility is consistent with the observation that in the absence of TNF, LPS can activate the expression of various genes, including some cytokine genes, in monocytes (27), macrophages (28), and TNF-{alpha}-deficient mice (29).

With respect to signaling pathways involved in the differentiation process, we found an association between DC differentiation and activation of NF-{kappa}B. All three inducing agents, LPS, TNF, and CI, resulted in nuclear NF-{kappa}B, and treatment that blocked the appearance of nuclear NF-{kappa}B also inhibited differentiation. Because it is well known that both TNF-{alpha} and LPS activate NF-{kappa}B in many cell types, these results are not surprising. In fact, the involvement of NF-{kappa}B, and especially RelB, in the process of LPS-induced DC differentiation has been reported previously (30, 31, 32). As in other cell types, signaling most likely proceeds through a Toll-like receptor (for LPS) or TNFR p55 (for TNF) through pathways that lead to the activation of the I{kappa}B kinases and hence to NF-{kappa}B (33, 34).

However, our observation that CI also activates NF-{kappa}B in the absence of any apparent costimulator was unexpected. There are many reports showing that Ca2+ synergizes with various other agents or treatments in activation of NF-{kappa}B, but we are unaware of any previous experiment showing that CI by itself can induce NF-{kappa}B. We were unable to exclude the possibility that GM-CSF supplies additional signals necessary for CI-induced activation of NF-{kappa}B because this cytokine was essential for maintaining cell viability. It also remains a possibility that CI acts indirectly, through the induced secretion of biological agents that, in turn, act in an autocrine fashion to promote differentiation. However, thapsigargin, a compound that causes a rapid efflux of Ca2+ from the endoplasmic reticulum, has been shown, as a single agent, to activate NF-{kappa}B in HeLa cells (35), normal renal tubular epithelial cells (36), and pancreatic lobules (37). This activation is inhibited by pyrrolidine dithiocarbamate and therefore requires the presence of reactive oxygen intermediates. We tried to test whether reactive oxygen intermediates were necessary for ionophore-induced NF-{kappa}B activation under our conditions, but unfortunately both pyrrolidine dithiocarbamate and NAC were very toxic for monocytes and HL-60 cells when used in combination with ionophore. Therefore, beyond the fact that it is CsA sensitive (and thus likely to involve calcineurin), the mechanism of ionophore-induced NF-{kappa}B activation and DC differentiation remains to be defined.

What is the role of NF-{kappa}B in the DC differentiation process? Many of the genes up-regulated in DCs contain functional NF-{kappa}B binding sites in their regulatory regions. These include the genes for IL-1{alpha}, IL-1ß, IL-6, IL-8, IL-12 (p40), macrophage inflammatory protein-1{alpha}, macrophage inflammatory protein-1ß, CCR5, CD80, MHC class I (H-2Kb), MHC class II (HLA-B7), CD54, Fas ligand, Fas, RANTES, and, of course, TNF-{alpha} (5, 38). In addition, the CD86 gene has a required NF-{kappa}B binding site (39), and the human CD83 gene has a potential NF-{kappa}B binding site whose functional significance has not yet been reported (40). Thus, expression of many genes important for DC function is likely to involve NF-{kappa}B. Expression of the pro-survival Bcl-XL gene is also up-regulated in DC and is influenced by NF-{kappa}B (41, 42), consistent with the anti-apoptotic function of RelA that has been demonstrated in many cell types. It is interesting, in light of the importance of RelB to DC function (see below), that RelB is also capable of inhibiting apoptosis in at least one model system (our unpublished observations).

Targeted disruption of the RelB gene in mice has revealed that it plays a unique and critical role in DC differentiation and/or function (21, 43). In wild-type mice, RelB is expressed largely in the thymus, lymph nodes, and spleen, and its localization in those organs correlates with that of DC. RelB-/- mice produce no apparent mature DC of myeloid origin, and bone marrow chimeras (RelB -/- bone marrow into lethally irradiated wild type host) have shown that this is due to a direct effect of RelB on stem cell development (21, 44). Consistent with this finding, we and others have shown that RelB is up-regulated and activated during differentiation of DC in vitro from both mouse and human precursors (19, 30, 45, 46). In this report we demonstrated two forms of the RelB protein. Whether these two forms differ in functional properties is a subject of our current studies. Most interestingly, we showed that RelB can be induced in myeloid cells either directly or indirectly through a calcium-dependent, CsA-sensitive pathway. In contrast, NF-{kappa}B/RelB activation induced by LPS or TNF-{alpha} was not sensitive to CsA, indicating that at least two different signaling pathways are available for the induction of NF-{kappa}B proteins and DC differentiation, each with distinct upstream components.


   Acknowledgments
 
We express our gratitude to Drs. Susan Leitman, E. J. Read, Harvey Klein, Thomas Trischmann, and Charles Carter of the Cell Processing Section of the Transfusion Medicine Branch at the National Institutes of Health for their generosity and technical support, as well as to Carol Shawver for kind assistance in preparing this manuscript.


   Footnotes
 
1 L.L. and G.K. contributed equally to this manuscript and share first authorship. Back

2 Address correspondence and reprint requests to Dr. Nancy R. Rice, Frederick Cancer Research and Development Center, National Cancer Institute, P.O. Box B, Frederick, MD 21702-1201. Back

3 Abbreviations used in this paper: DC, dendritic cell; CI, calcium ionophore; CsA, cyclosporin A; M{phi}-SFM, macrophage serum-free medium; NAC, N-acetyl cysteine; poly(dI-dC), polydeoxyinosinic-deoxycytidylic acid; TNFR1, TNF receptor 1. Back

Received for publication May 1, 2000. Accepted for publication July 14, 2000.


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