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Evidence for Distinct Intracellular Signaling Pathways in CD34⁺ Progenitor to Dendritic Cell Differentiation from a Human Cell Line Model¹

Daniel C. St. Louis^*,, Juliana B. Woodcock^*, Guido Fransozo, Patrick J. Blair^*, Louise M. Carlson, Maria Murillo^*, Mark R. Wells^*, Amanda J. Williams^*, Douglas S. Smoot^*, Sumesh Kaushal^*,, Janelle L. Grimes^*, David M. Harlan^*,§, John P. Chute^*,§, Carl H. June^*,§, Ulrich Siebenlist and Kelvin P. Lee^2,*,§

^* Immune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889; The Henry M. Jackson Foundation for the Advancement of Military Medicine, U.S. Military HIV Research Program, Bethesda, MD 20889; Laboratory of Immunoregulation, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892; and ^§ Department of Internal Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20889

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34⁺ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-) CD34⁺ HPCDC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34⁺ HPCDC differentiation we have characterized a human leukemic cell line model of this process. The CD34⁺ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-, or PMA (with or without the calcium ionophore ionomycin, or TNF-), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I⁺/MHC II^bright/CD83⁺/CD86⁺/CD14^- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms , ßI, ßII, and µ; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34⁺ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
How multipotential hemopoietic stem cells become mature blood cells is a paradigm of cellular differentiation and lineage commitment. Complex hemopoiesis underlies the generation of each component of the immune system, including T, B, and professional APCs (such as monocyte/macrophages and dendritic cells (DC)³). Evidence now suggests that DC play the central and critical role in both initiating and controlling the primary immune response via cognate interactions with T and B cells 1 . In humans, dendritic cells are a heterogeneous population shown in vitro to arise from multiple distinct hemopoietic progenitor cells (HPC) along distinct differentiation pathways. These progenitors include CD14⁺ monocytes 2, 3, 4, 5 and CD34⁺ bone marrow cells (in humans a heterogeneous population of multipotential progenitors of which the true pluripotent stem cell is a subset). CD34⁺CD10⁺ HPC give rise to the lymphoid DC lineage 6 , whereas CD34⁺CD86⁺ HPC are progenitors for myeloid DC and monocyte/macrophages 7 .

Broadly characterized from a cellular standpoint, there are two stages of DC differentiation 8 . The first stage involves differentiation of multipotential HPC to immature (or unactivated) DC, cells with high capacity for Ag uptake but relatively poor ability to activate T cells. This differentiation is induced in vitro by exogenous cytokines (GM-CSF and TNF- (with or without other cytokines) for CD34⁺ HPC 9 , and GM-CSF and IL-4 for monocytes 2, 3, 4, 5), CD40 receptor cross-linking (CD34⁺ HPC) 10 , or calcium ionophore alone (monocytes) 11 . The second stage involves maturation of immature to mature DC, cells that have decreased Ag uptake capability but are much more potent in activating T cells. An alternative viewpoint is that DC maturation is actually activation of resting DC. In vivo this second stage is probably triggered by "danger signals," which may include live bacteria and components (LPS, DNA), viral infection, and inflammatory cytokines (reviewed in 1 . In vitro, maturation/activation can be induced by a number of stimuli, including cytokines (TNF- with or without other cytokines, reviewed in 12 , monocyte-conditioned medium 13 , or CD40 receptor cross-linking 10 .

We have previously reported that the phorbol ester PMA alone induces differentiation of human CD34⁺ HPC into mature and fully functional DC, suggesting that direct activation of protein kinase C (PKC) by itself is sufficient to trigger this lineage commitment 14 . PMA-driven CD34⁺ HPCDC differentiation is specific for DC (no other lineages are generated) and does not involve proliferation (differentiation is complete within 7 days), although about 50% of the input number of CD34⁺ HPC are lost (in part via apoptosis). All these characteristics distinguish PMA from receptor-mediated differentiation signals (cytokines, CD40 receptor cross-linking). Since exogenous stimuli (including TNF-, IL-1ß, IL-4, and CD40) 15, 16, 17, 18, 19 used to induce CD34⁺HPCDC differentiation also activate PKC as part of their downstream signaling cascade, it is likely that PMA is activating the component of this cascade that specifically initiates DC differentiation. Consistent with this, we now report that inhibition of PKC activation suppresses generation of DC by GM-CSF and TNF- stimulation of CD34⁺ HPC.

Aside from a potential role for PKC activation, relatively little is known about what intracellular signaling pathways (and the genetic programs they initiate) are involved in HPCDC differentiation. Signaling via intracellular calcium flux alone (by calcium ionophore) induces monocyteDC differentiation 11 , although we have not observed this for CD34⁺ HPC (D.C.S.L and K.P.L, unpublished observations). The downstream components involved in this calcium signaling are uncharacterized. Another signaling pathway involves the lipid second messenger ceramide, which mediates down-regulation of Ag uptake during cytokine-driven maturation/activation of DC 20 . Downstream of these signaling pathways, the transcription factor RelB (a member of the NFB family) appears to play an important role in DC differentiation based on apparent function 21, 22, 23, 24 and the phenotype of RelB knockout mice 25, 26 . RelB expression is up-regulated in vitro during cytokine- or PMA-driven DC differentiation 2, 14 . What genes RelB specifically regulates during DC differentiation is currently unknown.

Even using agents that directly activate signal transduction pathways, a number of obstacles makes study of the intracellular and genetic events involved in DC differentiation difficult. The rarity of CD34⁺ HPC (0.1–1% of bone marrow mononuclear cells) makes sufficient isolation for larger scale studies laborious. In addition, CD34⁺ HPC are a heterogeneous population of progenitors already committed to different lineages (for example, see 6 , which makes analysis of bulk starting populations problematic. Receptor-mediated CD34⁺ HPCDC differentiation involves cell proliferation, which complicates identification of differentiation-specific processes. These stimuli also generate mixed populations of cells (DC, monocytes, neutrophils) that require additional purification of DC before definitive analysis (reviewed in 12 . Finally, CD34⁺ HPC have traditionally been difficult to transfect/transduce 27 , making genetic manipulation involving these approaches less effective.

Many of these problems could be circumvented in a cell line model of DC differentiation. Czerniecki et al. have suggested that the promyelocytic HL-60 cell line differentiates into DC with calcium ionophore, thus representing a potential model of monocyteDC differentiation 11 . It has recently been reported that committed CD34⁺ precursors for myeloid DC and macrophages are CD86 positive 7 . We have found that the CD34⁺ human myeloblastic cell line KG1 28 also expresses CD86. KG1 is a cytokine-responsive human CD34⁺ myelomonocytic cell line derived from a patient with erythroleukemia. KG1 was originally described to differentiate to the monocyte/macrophage lineage (based on morphologic and histochemical assays) when stimulated with phorbol ester 29, 30, 31 , although DC-specific markers were not available at that time. The KG1a subline of KG1 was indifferent to the effects of PMA (and cytokines) and is characterized as less differentiated than KG1 29, 30, 32 . KG1 and KG1a have qualitative differences in PKC activation and substrate phosphorylation 30, 33, 34 . We now report that phorbol ester or cytokine (GM-CSF and TNF-) induces KG1 to differentiate into dendritic-like cells (DLC) based on morphology, surface Ag phenotype, function, and gene expression. In contrast, KG1a do not differentiate. Characterization of stimuli required to induce DLC differentiation as well as comparison to KG1a provide evidence that distinct intracellular signaling pathways are involved and mediate specific components of differentiation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and culture

CD34⁺ HPC were isolated from organ donor bone marrow by immunomagnetic selection as previously described 35 . Purified cells were typically >95% CD34⁺ as determined by FACS analysis with a second noncross-blocked anti-CD34 mAb. For generation of DC, CD34⁺ HPC were cultured for 14 days at 5 x 10⁴ cells/ml in complete culture medium (IMDM, Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (HyClone, Logan UT), 100 mM L-glutamine, and 100 U/ml penicillin/streptomycin solution (Life Technologies)) with GM-CSF (200 U/ml) and TNF- (2.5 ng/ml; all from R&D Systems, Minneapolis, MN). Where indicated, staurosporine (Calbiochem, San Diego, CA) was added at 0.1 ng/ml, a dose sufficient to block PMA-induced proliferation of human T cells. At the end of the culture period, nonadherent cells and adherent cells were resuspended (3 mM EDTA), washed, and concentrated by centrifugation.

KG1 and KG1a cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in IMDM/20% FCS/100 mM L-glutamine/100 U penicillin/streptomycin at 5 x 10⁴ cells/ml. Cultures were stimulated alone or in combination with PMA (10 ng/ml; Sigma, St. Louis, MO), GM-CSF (200 U/ml), TNF- (10 ng/ml), ionomycin (100 ng/ml; Calbiochem). The four-cytokine combination of GM-CSF (2 ng/ml), IL-3 (5 ng/ml), IL-6 (5 ng/ml), stem cell factor (SCF; 120 ng/ml) has previously been found to induce robust proliferation of primary CD34⁺ HPC 35 . Where indicated, bisindolylmaleimide I (bis; Calbiochem) was used at 5 µM.

Flow cytometric analysis and mAbs

Adherent and loosely adherent cells were harvested with 3 mM EDTA, washed twice, and resuspended in staining medium (PBS, 5% FCS, 2% BSA, and 0.1% sodium azide). Phenotypic analysis of cells (2 x 10⁵) was performed by flow cytometry using saturating concentrations of the following mAbs: CD1a (clone SK9), CD2 (S5.2), CD4 (SK3), CD13 (L138), CD14 (MOP9), CD34 (8G12), CD80 (L307.4), and CD154 (89-76; all from Becton Dickinson, San Jose, CA); CD10 (HI10A), CD33 (WM53), CD40 (5C3), CD45RA (HI100), CD86 (IT2.2), and CD90 (5E10; all from PharMingen, San Diego, CA); CD83 (HB15, Immunotech, Westbrook, ME); MHC class I (H58A) and MHC class II (H42A, both from Veterinary Medical Research Development, Pullman, WA); c-fms(3-4A4), TNF receptor 1 (N-20), and TNF receptor 2 (C-20; all from Santa Cruz Biotechnology, Santa Cruz, CA). Appropriate conjugated isotype-matched Abs were used as controls. Ten thousand cells were analyzed on a Coulter Elite flow cytometer (Coulter, Hialeah, FL) through a viable cell gate as determined by forward and right angle light scatter parameters to exclude subcellular particles.

Apoptotic cells were identified by annexin V staining as previously described 36 . Briefly, cells were washed twice with cold PBS and then resuspended in a binding buffer (from the manufacturer) containing HEPES with 2.5 mM CaCl₂ at a concentration of 1 x 10⁶/ml. One hundred microliters of this suspension was reacted with 10 µl of annexin V-FITC (10 µg/ml (R&D Systems)) and 10 µl of propidium iodide reagent (50 µg/ml in PBS). The mixture was gently vortexed and then incubated for 15 min at room temperature in the dark. Following incubation, 400 µl of the binding buffer (1x concentration) was added to each tube. Analysis by FACS was conducted within 1 h of assay completion for optimal results. Controls of unreacted cells, propidium iodide-stained cells, and annexin-V-FITC-stained cells were run first to optimize settings.

FITC-dextran uptake

Analysis of micropinocytosis by FITC-dextran uptake has been previously described 37 . Activated KG1 and KG1a cells were resuspended at 10⁶/ml in IMDM/20% FCS. FITC-dextran (Sigma) was added at a final concentration of 1 mg/ml, and the mixture was incubated at either 4 or 37°C for 30 min. The cells were washed four times with cold PBS containing 1% FCS and analyzed by flow cytometry.

T cell activation

Human PBMC were obtained by leukapheresis from normal healthy adult donors. T cells were purified by negative selection as previously described 38 and resuspended in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 20 mM HEPES. T cells (1 x 10⁵/well) were cultured in the absence (in medium) or the presence of the indicated number of gamma-irradiated (3000 rad ¹³⁷Cs), in vitro-generated DC or DLC. Cultures were incubated for 3 days (DC) or 5 days (DLC) at 37°C in a humidified 5% CO₂ in air atmosphere. T cell proliferation induced by freshly isolated allogeneic PBMC was used as a reference. Proliferation was assessed after the addition of 0.5 µCi/well [methyl-³H]thymidine (New England Nuclear, Boston, MA) for the final 24 h of culture. Cells were harvested using a 96-well cell harvester, and [methyl-³H]thymidine incorporation was measured using a Beta Plate scintillation counting system (Pharmacia/LKB, Gaithersburg, MD). All determinations were performed in triplicate and expressed as the mean counts per minute ± 1 SD.

RT-PCR and Northern blot analysis

Total RNA was isolated from cultured cells by the RNA STAT-60 (Tel-Test) according to manufacturer’s recommendations. Ten micrograms of purified RNA was incubated at 37°C for 15 min with 100 U of RNase-free DNase in a 50-µl reaction, followed by phenol/chloroform extraction and ethanol precipitation. Samples of 4 µg of DNase-treated RNA were reverse transcribed to cDNA by random primer extension in the presence of 0.1 µCi of [-³²P]dCTP using Superscript-II Reverse Transcriptase (Life Technologies) following the manufacturer’s instructions. The RT reaction was terminated by heating at 70°C for 5 min, and radiolabeled cDNA was then purified on Spin-50 columns (International Mould Engineering, Odenton, MD) by centrifugation at 3500 rpm for 3 min. To normalize the input in all cDNA samples, equal amounts of cDNA counts were loaded in each PCR reaction containing 0.5 µM gene-specific primer pairs. Oligonucleotide primers specific for human DC-CK1 (sense, AGT CCC ATC TGC TAT GCC CAG; antisense, TAC GAA GAG TGG AAG GGA AAG) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense, ATG GGG AAG GTG AAG GTC GGA GTC AAC GGA; antisense, AGG GGG CAG AGA TGA TGA CCC TTT TGG CTC) were used. PCR was performed at 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 30 cycles followed by a final extension time of 10 min at 72°C. The amplified products (15 µl) were then incubated at 94°C for 5 min with 1 x 10⁵ cpm of purified [-³²P]dATP-labeled gene-specific probes followed by an incubation at 55°C for 10 min. Hybridized PCR products were then resolved on 6% PAGE followed by quantitation on a PhosphorImager scanner using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Northern blot analysis was performed as previously described 39 . Briefly, total RNA from unstimulated and stimulated KG1 and KG1a cells was isolated at the times indicated and equalized by serial dilution and ethidium bromide visualization. Equal amounts of RNA were separated on a formaldehyde/agarose gel, transferred to nylon membranes, and hybridized against a human RelB cDNA probe. The same blots were then probed for actin to assess equal loading and RNA integrity.

Western blot analysis

Cell lysates were made from KG1 and KG1a cells cultured under the conditions and times indicated. Samples containing equal amounts of protein were separated by SDS-PAGE (4% stacking/7.5% resolving); electroblotted to nitrocellulose; probed with Abs against RelB and PKC, -ßI, -ßII, and -µ (all from Santa Cruz Biotechnology, Santa Cruz, CA); and visualized by chemiluminescent detection (ECL, Amersham, Aylesbury, U.K.).

Electromobility shift assays

Cell lysates were made from KG1 and KG1a cells cultured under the conditions and times indicated. Samples containing equal amounts of protein were incubated with a ³²P-labeled oligonucleotide primer containing multiple NFB binding sites with or without anti-RelB Ab or anti-NFB (p50). Samples were then separated on a 4% polyacrylamide gel and exposed to film.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of PKC activation blocks cytokine-driven CD34⁺ HPCDC differentiation

We have previously shown that PMA activation of PKC induces human CD34⁺ HPCDC differentiation, which could be blocked by the PKC inhibitor staurosporine. To assess whether cytokine-induced CD34⁺HPCDC differentiation also involves PKC intracellular signaling, we cultured CD34⁺ HPC in GM-CSF and TNF- for 14 days with or without 0.2 nM staurosporine, a dose 3.5-fold less than the IC₅₀ of PKC (0.7 nM; Calbiochem Technical Report) but at least 10- to 200-fold lower than the IC₅₀ for protein kinase A and tyrosine kinases 40 . Cell viability at 14 days was >90% in both culture conditions, but there was a greater expansion of cell number without staurosporine (3x input number vs 1x with staurosporine). As can be seen in Fig. 1A, CD34⁺ HPC cultured in cytokine alone developed characteristic DC morphology (loosely adherent large cells with irregular shape, prominent dendritic processes, and hair-like cytoplasmic projections). In contrast, cultures containing staurosporine lacked cells with neurite processes and had greatly reduced numbers with hair-like cytoplasmic projections.

View larger version (83K): [in this window] [in a new window]   FIGURE 1. Primary CD34+ HPC cultured for 14 days in GM-CSF (200 U/ml) and TNF- (2.5 ng/ml) with or without staurosporine (0.2 nM). A, Morphology. Photomicrograph of the cultures. Black dots are magnetic beads used in the initial CD34+ purification. Arrows point to the characteristic neurite processes. B, Surface Ag phenotype. CD34+ HPC were cultured as described above, and adherent and nonadherent cells were analyzed by flow cytometry with the Abs indicated (solid line) vs isotype control (dotted line). The x-axis is log fluorescence, and the y-axis is cell number. C, Induction of allogeneic T cell proliferation. CD34+ cells cultured with or without staurosporine as described above were harvested on day 14, washed three times in PBS, plated in triplicate in the numbers indicated, and gamma irradiated (3000 rad 137Cs). Allogeneic irradiated PBMC were used for comparison. Purified CD4+ T cells (1 x 105) were added to each well. T cells were purified from leukapheresis collections of healthy donors by negative selection (38) and were free of accessory cells as demonstrated by Con A unresponsiveness. After 54 h of culture, cells were pulsed with 0.5 µCi/well [methyl-3H]thymidine before being harvested at 72 h. [3H]TdR incorporation was measured using a Beta Plate scintillation counting system.

Given that DC are the most effective APC in activating T cells, the decreased numbers of DC in the staurosporine-treated cultures should be reflected in a similar decrease in functional potency. CD34⁺ HPC cultured in GM-CSF and TNF- with staurosporine were significantly less effective in inducing allogeneic T cell proliferation than those cultured in cytokine alone, particularly at a low APC/T cell ratio (Fig. 1C). However, both were still superior to an unselected population of PBMC.

Together these data suggest that inhibition of PKC activation preferentially suppressed generation of DC from CD34⁺ HPC by GM-CSF and TNF-, findings consistent with the differentiating effects of phorbol ester. However, the scarcity, heterogeneity, and difficulty of manipulating primary CD34⁺ HPC made further analysis of the relevant signaling and genetic events difficult. To overcome these obstacles we sought to establish a human cell line model of CD34⁺ HPCDC differentiation. Although a heterogeneous lineage, DC share a constellation of defining characteristics, including morphology, lineage markers, function, and gene expression, that can be used to identify potential DLC.

KG1 develop characteristic DC phenotype (morphology, surface Ag expression) in response to cytokines or PMA

We first examined the PMA-responsive CD34⁺ myeloblast cell line KG1. KG1 cells in medium alone were typically round nonadherent cells (Fig. 2). When cultured in GM-CSF and TNF- (with or without IL-4), PMA or PMA plus TNF- (and PMA plus ionomycin; not shown), KG1 became loosely adherent and a subset developed long neurite processes and hair-like cytoplasmic projections (best seen by Wright staining; not shown), morphology reported to be characteristic of DC 12 . Morphologic changes began within 30 min of stimulation, were fully manifested within 48 h, and were stable over at least 2 wk in culture. Development of the dendrite/neurite morphology was progressively more prevalent and pronounced with GM-CSF plus TNF-PMAPMA plus TNF- (and PMA plus ionomycin). In contrast to these stimuli, there was no effect of individual cytokines (GM-CSF, IL-4, or TNF-), a cytokine combination that does not induce DC differentiation (GM-CSF, IL-3, IL-6, and SCF), ionomycin alone, or ionomycin plus TNF- (data not shown). While KG1a also became adherent when similarly stimulated, they did not change morphology.

View larger version (155K): [in this window] [in a new window]   FIGURE 2. Morphology of KG1 stimulated with cytokines and/or PMA. Photomicrographs of day 7 cultures of KG1 in medium alone, GM-CSF (200 U/ml) plus TNF- (2.5 ng/ml), PMA (10 ng/ml), or PMA plus TNF- (10 ng/ml). Arrows point to neurite processes. KG1 in medium alone were nonadherent; in all other conditions they were adherent.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. KG1 and KG1a expression of TNF- receptors 1 and 2. Unstimulated KG1 and KG1a were analyzed by FACS for TNF receptor 1 and 2 expression as described in Materials and Methods. The isotype-matched control is the dotted line, and Ag-specific Ab the solid line.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. Surface Ag phenotypes of KG1 and KG1a in response to cytokine and/or PMA stimulation. KG1 cells were cultured in medium alone, GM-CSF (200 U/ml) plus TNF- (2.5 ng/ml), PMA (10 ng/ml), PMA plus TNF- (10 ng/ml), or PMA plus ionomycin (200 ng/ml). KG1a were cultured in PMA (10 ng/ml). Cells were harvested and analyzed by FACS. Ag-specific staining is indicated by the solid line, and the isotype-matched control is shown by the dotted line. The time of maximal surface expression varied depending on the stimulus, shown here are GM-CSF plus TNF- and PMA plus ionomycin (day 3 in culture), and medium, PMA, and PMA plus TNF- (day 7 in culture).

Signals that induce KG1DLC differentiation also suppress proliferation and trigger programmed cell death

In primary CD34⁺ HPC we found the two hallmarks of PMA-driven DC differentiation are the complete inhibition of proliferation (even to exogenous cytokines) and induction of apoptosis 14 . Proliferation is also absent in monocyteDC differentiation driven by GM-CSF, IL-4, and TNF- 4 or calcium ionophore 11 . As shown in Fig. 5, KG1 proliferation was suppressed by factors that induce DC/DLC differentiation (PMA (consistent with the findings presented in 44 , TNF-, and most significantly by PMA plus TNF-), but not by a cytokine combination (GM-CSF, IL-3, IL-6, and SCF) that does not generate DC. Baseline proliferation of KG1a was 2.6-fold higher than that of KG1 and was unaffected by these stimuli (data not shown). Because the day 5 viability of KG1 was decreased in PMA (75% viable), PMA plus TNF- (70%), and PMA plus ionomycin (67%), we examined the induction of apoptosis by these stimuli (Table II). In KG1, apoptosis was triggered within 4 h by PMA plus iono and within 24 h by PMA plus TNF-. Smaller increases in apoptotic cell numbers were seen at 24 h in cultures treated with PMA or TNF- alone, correlating with a lesser inhibition of cell proliferation by these single factors. Also consistent with the proliferation data was the failure of any stimuli to induce apoptosis in KG1a (the greater baseline percentage of apoptotic cells was probably due to the higher proliferative rate).

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Inhibition of KG1 proliferation. KG1 were cultured at the cell numbers and in the conditions noted. After 54 h of culture, cells were pulsed with 0.5 µCi/well [methyl-3H]thymidine before being harvested at 72 h. [3H]TdR incorporation was measured using a Beta Plate scintillation counting system.

Ag uptake (via receptor-mediated endocytosis (Fc, mannose receptors) or macropinocytosis of fluid phase molecules) is a functional characteristic of both DC and CD34⁺CD86⁺ DC precursors 7, 37 . Ag capture (as measured by FITC-dextran uptake) changes from a high capacity in immature/unactivated DC to a lower capacity in mature/activated DC 37 . Unstimulated KG1 took up FITC-dextran, and PMA did not affect uptake even though it induced other characteristic DC changes (Fig. 6). However, TNF- alone or in combination with PMA down-modulated macromolecule uptake, suggesting that TNF- (but not PKC activation) delivers a distinct signal that mediates the decreased Ag uptake capability associated with DC maturation.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Fluid phase macromolecule uptake by KG1 and KG1-DLC. KG1 were unstimulated (medium) or cultured in PMA, TNF-, or PMA plus TNF- for 5 days. FITC-dextran uptake was then assayed as described in Materials and Methods and analyzed by flow cytometry. FITC-dextran uptake at 4°C by unstimulated KG1 served as the negative control.

The most distinctive functional characteristic of DC is the potent ability to activate T cells 45 . We found that PMA-generated KG1 DLC were capable of stimulating allogeneic T cell proliferation (Fig. 7) and to a comparatively greater extent than unselected PBMC (as a reference population of APC). KG1 cultured in PMA and PMA plus ionomycin were significantly more potent at inducing T cell proliferation than KG1 cultured in medium, KG1a cultured in PMA with or without ionomycin, or unselected PBMC. Consistent with the morphology and surface phenotype findings above, ionomycin appeared to drive further maturation/activation over PMA alone as measured by alloproliferation. Lack of significant T cell proliferation with PMA-treated KG1a cells demonstrates the lack of PMA carryover from the original KG1/KG1a differentiation cultures.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. T cell proliferation in response to KG1 and KG1a cultured in a variety of conditions. KG1 and KG1a cells were cultured for 7 days in medium, ionomycin (50 ng/ml), PMA (10 ng/ml), and PMA plus ionomycin. Cells were thoroughly washed and added to purified resting allogeneic T cells in flat-bottom microtiter plates at an APC:T ratio of 1:10. Freshly isolated allogeneic PBMC were used as APC for comparison. Proliferation was measured by [3H]TdR incorporation.

Characterization of DC has classically relied on morphology, surface Ag expression, and function. Recent molecular studies have identified genes that are expressed predominantly or exclusively by DC. These include the RelB transcription factor (see below) and the chemokine DC-CK1. The DC-CK1 chemokine is expressed only by DC and not other APC, and appears to function as a chemoattractant for naive T cells 46 . Unstimulated KG1 expressed low levels of DC-CK1 that were substantially up-regulated by PMA (Fig. 8A). Unexpectedly, the addition of TNF- inhibited DC-CK1 up-regulation in KG1. The converse response was seen in KG1a (Fig. 8B), where PMA has no effect on DC-CK1 expression but PMA plus TNF- results in up-regulation, arguing against a direct inhibitory effect of TNF-. Since PMA plus TNF- induces early DLC differentiation in KG1a (based on CD83 expression), these findings suggest that DC-CK1 expression is maximal during early/intermediate stages of DC maturation/activation (induced by PMA in KG1, PMA plus TNF- in KG1a) with much less expression in uncommitted progenitors (KG1a) or fully mature/activated DC (PMA- plus TNF--treated KG1).

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Up-regulation of DC-CK1 expression during PMA-driven KG1DLC differentiation. KG1 and KG1a cells were stimulated with PMA with or without TNF-, and DNase-treated RNA were made at the times indicated. Equal amounts of cDNA from mRNA were amplified by PCR with gene-specific primers, hybridized to internal DC-CK 1-specific oligonucleotide, separated by PAGE, exposed, and visualized on a phosphorimager. Expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same samples was used as a control.

Role of PKC in PMA-driven KG1DLC differentiation

Although PMA is a stable 2,3-diacylglycerol analogue and specific activator of the classical and new isoforms of PKC 47 , it is formally possible that PMA’s effects in KG1 are due to activation of a non-PKC phorbol ester receptor (such as Unc-11 or n-chimaerin) 48, 49, 50 . To more rigorously prove specific PKC activation, KG1 were cultured in PMA with or without TNF- with the highly selective PKC inhibitor bis. Bis inhibited adhesion and morphologic changes in KG1 cultured in PMA without affecting cell viability (Fig. 9A). By FACS, bis inhibited up-regulation of CD83 and CD86 in response to both PMA and PMA plus TNF- (Fig. 9B). Bis also prevented cell death induced by PMA plus TNF- (91% viable with PMA, TNF-, and bis on day 5 vs 37% viable with PMA plus TNF- alone). Finally, the addition of bis inhibited the development of allostimulatory capacity in KG1 cultured in PMA or PMA plus ionomycin (Fig. 9C). The fact that bis inhibition is considerably less effective with the addition of ionomycin is probably due to a lowered PKC activation threshold caused by increased intracellular calcium.

View larger version (44K): [in this window] [in a new window]   FIGURE 9. The effect of the PKC inhibitor bis on PMA-driven KG1DLC differentiation. KG1 and KG1a cells were cultured for 7 days in medium; bis (1 µM); PMA (10 ng/ml); PMA plus bis; PMA plus TNF- (10 ng/ml); PMA, TNF-, and bis; PMA plus ionomycin (50 ng/ml); and PMA, ionomycin, and bis. A, Morphology. Photomicrographs of KG1 cultured in PMA or PMA plus bis are shown. The focal plane is the bottom of the well (adherent cells). Arrows point to characteristic neurite processes. B, Surface Ag phenotype. Ten thousand day 7 cells were analyzed as described in Materials and Methods. Dotted lines show the isotype control; solid lines show the Ag-specific Ab. C, Induction of allogeneic T cell proliferation. KG1 and KG1a were cultured as indicated, thoroughly washed, and added to purified resting allogeneic T cells in flat-bottom microtiter plates at an APC:T ratio of 1:10. Freshly isolated PBMC were used for comparison. Proliferation was measured by [3H]TdR incorporation. The data in this figure are a subset of those for the experiment presented in Fig. 7.

View larger version (29K): [in this window] [in a new window]   FIGURE 10. Western blot analysis of PKC isoform expression in KG1 and KG1a cells. Cell lysates were made from unstimulated KG1 and KG1a cells. Samples containing 20 µg of protein were separated by SDS-PAGE (5% stacking/10% resolving), electroblotted to nitrocellulose, probed with Abs against specific PKC isoforms, and visualized by chemiluminescent detection. The same results were obtained using equal numbers of cells per lane.

Induction of RelB expression during PMA-driven KG1DLC differentiation

RelB is a member of the NFB transcription factor family that is highly expressed in DC but not monocytes 2 and may play an important role in DC differentiation and function. Because up-regulation/activation of RelB by PMA may represent the pathway by which PKC-mediated signaling gains access to the nucleus, we examined the kinetics of RelB expression following PMA activation of KG1 and KG1a. As can be seen in Fig. 11A, RelB gene expression in KG1 was up-regulated within 24 h, plateaued through 72 h, and then declined. Unexpectedly, unstimulated KG1a constitutively expressed RelB message, which also declined after 72 h in culture with PMA. Protein expression paralleled the inducible KG1 or constitutive KG1a gene expression of RelB (Fig. 11B). Of note is the rapidity with which RelB protein was induced in KG1, appearing within 30 min of PMA stimulation. The persistence of RelB protein after gene down-regulation suggests a relatively slow turnover rate. By electromobility shift assays the levels of free (i.e., not bound to IB) RelB heterodimers capable of binding to NFB sites were also up-regulated by PMA in KG1, while levels in KG1a were comparatively stable (Fig. 11C, upper panels). Examination of other NFB family members in KG1 and KG1a revealed that nearly all protein capable of binding NFB sites contained p50 (NFBI) as one subunit (Fig. 11C, lower panels) as evidenced by the nearly complete supershifting by anti-p50 Abs. This suggests that in KG1/KG1a RelB exists as a RelB/p50 heterodimer.

View larger version (31K): [in this window] [in a new window]   FIGURE 11. Expression of RelB in PMA-stimulated KG1 and KG1a. KG1 and KG1a were cultured in 10 ng/ml PMA for the times indicated. A, RelB mRNA expression. Cells were harvested and mRNA isolated as described in Materials and Methods. Total mRNA was equalized between samples by ethidium bromide visualization and serial dilution. Samples were separated on formaldehyde agarose gels, transferred to nylon, and probed with 32P-labeled RelB cDNA probe. The same blot was then reprobed for actin expression to verify mRNA equalization and integrity. B, RelB protein expression. Cell lysates from the indicated times points were made, and equal amounts of protein were loaded onto a SDS-PAGE gel and analyzed by Western blotting with the anti-RelB Ab C-19. C, RelB and p50 electromobility shift assay. Cell lysates were made at the times indicated. Equal amounts of protein were incubated with a labeled primer containing the NFB binding site with or without anti-RelB or anti-p50 mAb. Samples were separated on 4% polyacrylamide gel and exposed to film. Complexes that specifically contain the labeled primer, Rel B (or p50), and anti-RelB (or p50) Ab are indicated by the supershift arrow.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To further study the intracellular events involved in DC differentiation and to circumvent the problems of studying this in primary CD34⁺ HPC, we established a human leukemic cell line model of CD34⁺ HPCDC differentiation. Work demonstrating that primary chronic myelogenous leukemia cells differentiated in vitro to functional DC suggested that this was feasible 70 . Examination of the PMA responsiveness of a panel of CD34⁺ leukemic cell lines revealed that KG1 differentiated into cells with DC characteristics. KG1 was isolated from a patient with erythroleukemia (FAB M7) undergoing myeloblastic relapse 28 . Serial passage of KG1 gave rise to the KG1a subline 71 , which by a number of criteria (surface phenotype, gene expression, and growth factor unresponsiveness) is thought to be arrested at a less differentiated stage than KG1 32, 72, 73 . In response to PMA KG1 was initially characterized to differentiate into macrophages 74 , although these original morphologic (adherent cells with long pseudopodia), histochemical (nonspecific esterase-positive, myeloperoxidase-negative), surface Ag (MHC II- and Fc receptor-positive), and functional (phagocytosis) findings have since been found to also be characteristic of DC (reviewed in 12 . PMA does not induce differentiation in KG1a, and although KG1 and KG1a have similar numbers of phorbol ester binding sites, they differ in PKC activity, translocation, and substrate phosphorylation 30, 33, 34, 74 .

We believe that recharacterization of the KG1 response to PMA reveals it to actually recapitulate differentiation of the committed CD34⁺ myeloid DC/macrophage progenitor to DC. Like the committed DC/macrophage progenitor 7 , KG1 express MHC II, CD34, and CD86, whereas KG1a are MHC II^-CD34⁺CD86^-. Unstimulated KG1 express little or no CD40, but up-regulate expression following culture in PMA. Stimuli shown to induce CD34⁺HPCDC differentiation (GM-CSF plus TNF-, PMA) also induce differentiation of KG1 to DLC (KG1-DLC), while non-DC cytokine(s) (GM-CSF or TNF- alone or GM-CSF, IL-3, IL-6, and SCF) do not. Unlike monocytes, ionomycin alone does not induce DC differentiation in KG1 (or primary CD34⁺ HPC), and likewise, PMA (as a single agent) does not drive DC differentiation in monocytes. Suppression of KG1 cell proliferation and induction of apoptosis by PMA are also characteristics we reported for primary CD34⁺ HPCDC differentiation driven by PMA.

Stimulation of KG1 with GM-CSF and TNF- or PMA (with or without TNF- or ionomycin) generates cells with a constellation of DC characteristics. Morphologically, KG1-DLC are adherent with long neurite processes in culture and have hair-like processes/veils on cytospin. By surface Ag phenotype, KG1-DLC have characteristic DC expression of MHC I, MHC II (high), CD13, CD33, the DC-specific lineage marker CD83, and the costimulatory ligand CD86 (reviewed in 12 . They do not express CD1a or the monocyte marker CD14. Expression of CD80 is present but low, suggesting that KG1-DLC are not fully mature or activated 12 . Functionally, unstimulated KG1 can take up fluid phase Ag as measured by FITC-dextran uptake. TNF- appears to deliver a signal that down-regulates Ag uptake, a hallmark of DC maturation/activation 37 consistent with the other effects (morphology, surface Ag phenotype) that TNF- has on PMA-driven KG1DLC differentiation. Most importantly, KG1-DLC can function to activate T cells as evidenced by proliferation in an allo-MLR. This proliferation is substantially higher than that induced by either allogeneic PBMC or similarly cultured KG1a. It is interesting to note that even though unstimulated KG1 express MHC II and CD86, they do not stimulate significant T cell proliferation. Because MHC II and CD86 expression are not substantially up-regulated by PMA, this suggests the enhanced allostimulatory properties of KG1-DLC are due to other components not measured. Assessment of cytokine production (especially IL-12) is currently underway. Finally, KG1-DLC express genes that are predominantly (RelB) or exclusively (DC-CK1) found in DC. Together, these four levels of characterization all point to the conclusion GM-CSF plus TNF- or PMA stimulation of KG1 generates cells of the DC lineage.

The inability to induce DLC differentiation in KG1a allows for comparison with KG1 of potential signaling pathways. Blockade of DC differentiation in both KG1 and primary CD34⁺ HPC by PKC inhibitors establishes a central role for PKC. PKC activation most likely plays a direct role and does not simply induce autocrine secretion of cytokines; for example, we find no evidence (by PCR) for induction of TNF- expression in KG1. However, the difference between KG1 and KG1a cannot simply be the absence of PKC signaling in KG1a given that both cell lines have biochemically measurable PKC activity 75 . Likewise, we have found that although both CD34⁺ HPC and CD34^- HPC respond to PMA, only CD34⁺ HPC differentiate to DC while CD34^- HPC become macrophages. This suggests that there is a qualitative component of PKC activation. Consistent with this is the differing expression of specific PKC isoforms in the cell lines. KG1a express less PKC and no PKCßI, -ßII, or -µ compared with KG1. Given that specific PKC isoforms have been associated with specific biological events in other systems (e.g., PKC in T cell activation 51 and PKCß in macrophage differentiation of HL-60 52), these findings suggest that the same may be true for DC differentiation. Examination of the roles of specific PKC isoforms is currently underway.

KG1a also differ from KG1 in the response to TNF-; this is most evident in the induction of apoptosis. This difference may be due to comparatively lower expression of both TNF receptors 1 and 2 on KG1a, and this membrane "defect" is consistent with previous reports demonstrating that apoptotic responses in KG1a can be restored by directly manipulating the sphingomyelin-ceramide pathway triggered by TNF- 76, 77 . The roles of ceramide (and intracellular calcium flux) in both apoptosis and DC differentiation are under study.

In addition to the differences between KG1 and KG1a, we found that distinct signals appeared to mediate distinct, nonoverlapping biological phenomena during KG1DLC differentiation. PKC activation by PMA appears to be a requisite inductive signal, initiating morphology changes, surface Ag expression, and allostimulatory capacity for T cells. However, the addition of a TNF- or calcium flux signal is necessary for full maturation/activation (as assessed by morphology, surface Ag expression, and T cell proliferation) and inhibition of cell proliferation/induction of apoptosis. Down-regulation of soluble macromolecule uptake appears to be mediated entirely by TNF- and to be independent of PKC activation. The possibility that TNF- and ionomycin are signaling through the same pathway is suggested by the lack of an additive effect when the two are combined (PMA, TNF-, and ionomycin) and is being examined. Together, these data suggest that there are discrete signaling pathways involved in specific aspects of DC differentiation; PKC activation is required to initiate differentiation, and TNF- (which initiates several intracellular pathways) and/or intracellular calcium flux is required for terminal maturation/activation and apoptosis. However, this does not explain why some single signals (PMA or CD40 cross-linking in primary CD34⁺ HPC, calcium ionophore in monocytes) are capable of driving complete DC differentiation/maturation from progenitors. One possibility is that those particular pathways in those particular progenitors are directly linked to the genetic DC differentiation program. Another possibility is that the primary signal in these specific progenitors triggers autocrine secretion of additional cytokines/factors that drive terminal maturation. We are currently examining whether PMA induces TNF- expression or calcium flux in primary CD34⁺ HPC.

Regardless of the specific pathway, differentiation signals have to cross into the nucleus to initiate the requisite genetic programs. Such bridges are very often preformed transcription factors, of which the NFB family is one of the best characterized (reviewed in 78 . Critical involvement of NFB-responsive genes in DC differentiation has been found in chicken bone marrow cell lines 79 . Involvement of rel/NFB family transcription factors in DC differentiation is further suggested by the observations that RelB 80, 81 is expressed at high levels in murine DC 82 and that RelB knockout mice have significant reductions in mature DC number and APC function 83, 84 . In human DC nuclear localization of RelB is associated with enhanced APC function 85 . Both PMA and TNF- up-regulate the activity of NFB family members (including RelB by PMA) 86 , and RelB up-regulation is seen during CD34⁺ HPCDC 14 and monocyteDC 2 differentiation. This is also true for KG1DLC differentiation. However, constitutive expression of RelB in KG1a suggests that DC differentiation may not simply be due to activation of RelB. It is also possible that tonic expression of RelB somehow blocks induction of NFB-responsive genes as has been suggested for conditional v-rel expression 79 . It is unclear why RelB expression is not regulated in KG1a, but this suggests that some negative feedback signal has been lost. The rapid up-regulation of RelB expression induced by PMA in KG1 may indicate the involvement of a more immediate transcription factor, although NFB can mediate its own transcription 78 .

Together, the data presented in this report corroborate an important role for PKC activation in DC differentiation from CD34⁺ HPC. Characterization of the KG1 cell line model of CD34⁺HPCDC differentiation allows for a more detailed study of the signal transduction pathways involved. Our initial studies indicate that the PKC, TNF-, and/or intracellular calcium signaling pathways mediate specific phenomenon during this differentiation. A potential connection of these pathways to the RelB transcription factor may provide a doorway into uncovering the genes that control DC differentiation and underlie the unique function of this lineage.

	Footnotes

 
¹ This work was supported by the Naval Medical Research and Development Command, Research Task 62233N.MM33C30.1402.HD402.1412. Views presented in this paper are those of the authors; no endorsement by the Department of Navy, the Department of the Army, or the Department of Defense has been given or should be inferred.

² Address correspondence and reprint requests to Dr. Kelvin P. Lee, Immune Cell Biology Program, Immune Suppression Branch, Building 17, Room 214, Naval Medical Research Institute, 8901 Wisconsin Ave., Bethesda, MD 20889-5067. E-mail address:

³ Abbreviations used in this paper: DC, dendritic cell; HPC, hemopoietic progenitor cell; CD34⁺ HPC, CD34⁺ hemopoietic progenitor cell; GM-CSF, granulocyte-macrophage CSF; PKC, protein kinase C; DLC, dendritic-like cell; IMDM, Iscove’s modified Dulbecco’s medium; SCF, stem cell factor; bis, bisindolylmaleimide I.

Received for publication August 11, 1998. Accepted for publication December 11, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
2. Akagawa, K. S., N. Takasuka, Y. Nozaki, I. Komuro, M. Azuma, M. Ueda, M. Naito, K. Takahashi. 1996. Generation of CD1⁺ RelB⁺ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated giant cells from human monocytes. Blood 88:4029.[Abstract/Free Full Text]
3. Pickl, W. F., O. Majdic, P. Kohl, J. Stockl, E. Riedl, C. Scheinecker, C. Bello-Fernandez, W. Knapp. 1996. Molecular and functional characteristics of dendritic cells generated from highly purified CD14⁺ peripheral blood monocytes. J. Immunol. 157:3850.[Abstract]
4. Zhou, L. J., T. F. Tedder. 1996. CD14⁺ blood monocytes can differentiate into functionally mature CD83⁺ dendritic cells. Proc. Natl. Acad. Sci. USA 93:2588.[Abstract/Free Full Text]
5. Kiertscher, S. M., M. D. Roth. 1996. Human CD14⁺ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4. J. Leukocyte Biol. 59:208.[Abstract]
6. Galy, A., M. Travis, D. Cen, B. Chen. 1995. Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity 3:459.[Medline]
7. Ryncarz, R. E., C. Anasetti. 1998. Expression of CD86 on human marrow CD34(+) cells identifies immunocompetent committed precursors of macrophages and dendritic cells. Blood 91:3892.[Abstract/Free Full Text]
8. Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, P. Ricciardi-Castagnoli. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185:317.[Abstract/Free Full Text]
9. Caux, C., C. Dezutter-Dambuyant, D. Schmitt, J. Banchereau. 1992. GM-CSF and TNF- cooperate in the generation of dendritic Langerhans cells. Nature 360:258.[Medline]
10. Flores-Romo, L., P. Bjork, V. Duvert, C. Van Kooten, S. Saeland, J. Banchereau. 1997. CD40 ligation on human cord blood CD34⁺ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells. J. Exp. Med. 2:341.
11. Czerniecki, B. J., C. Carter, L. Rivoltini, G. K. Koski, H. I. Kim, D. E. Weng, J. G. Roros, Y. M. Hijazi, S. Wu, S. A. Rosenberg, et al 1997. Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. J. Immunol. 159:3823.[Abstract]
12. Hart, D. N.. 1998. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
13. Reddy, A., M. Sapp, M. Feldman, M. Subklewe, N. Bhardwaj. 1997. A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 90:3640.[Abstract/Free Full Text]
14. Davis, T. A., A. A. Saini, P. J. Blair, B. L. Levine, N. Craighead, D. M. Harlan, C. H. June, K. P. Lee. 1998. Phorbol esters induce differentiation of human CD34⁺ hemopoietic progenitors to dendritic cells: evidence for protein kinase C-mediated signaling. J. Immunol. 160:3689.[Abstract/Free Full Text]
15. Arruda, S., J. L. Ho. 1992. IL-4 receptor signal transduction in human monocytes is associated with protein kinase C translocation. J. Immunol. 149:1258.[Abstract]
16. Harnett, M. M., M. J. Holman, G. G. Klaus. 1991. IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down- regulation in murine B cells. J. Immunol. 147:3831.[Abstract]
17. Finney, M., G. R. Guy, R. H. Michell, J. Gordon, B. Dugas, K. P. Rigley, R. E. Callard. 1990. Interleukin 4 activates human B lymphocytes via transient inositol lipid hydrolysis and delayed cyclic adenosine monophosphate generation. Eur. J. Immunol. 20:151.[Medline]
18. Schutze, S., T. Machleidt, M. Kronke. 1994. The role of diacylglycerol and ceramide in tumor necrosis factor and interleukin-1 signal transduction. J. Leukocyte Biol. 56:533.[Abstract]
19. Ren, C. L., S. M. Fu, R. S. Geha, T. Morio. 1994. Protein tyrosine kinase activation and protein kinase C translocation are functional components of CD40 signal transduction in resting human B cells: signal transduction via CD40 involves activation of Lyn kinase and phosphatidylinositol-3-kinase, and phosphorylation of phospholipase C2. J. Exp. Med. 179:673.[Abstract/Free Full Text]
20. Sallusto, F., C. Nicolo, R. De Maria, S. Corinti, R. Testi. 1996. Ceramide inhibits antigen uptake and presentation by dendritic cells. J. Exp. Med. 184:2411.[Abstract/Free Full Text]
21. Carrasco, D., R. P. Ryseck, R. Bravo. 1993. Expression of RelB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells. Development 118:1221.[Abstract]
22. Boehmelt, G., J. Madruga, P. Dorfler, K. Briegel, H. Schwarz, P. J. Enrietto, M. Zenke. 1995. Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-RelER. Cell 80:341.[Medline]
23. Feuillard, J., M. Korner, A. Israel, J. Vassy, M. Raphael. 1996. Differential nuclear localization of p50, p52, and RelB proteins in human accessory cells of the immune response in situ. Eur. J. Immunol. 26:2547.[Medline]
24. Lernbecher, T., B. Kistler, T. Wirth. 1994. Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. EMBO J. 13:4060.[Medline]
25. Burkly, L., C. Hession, L. Ogata, C. Reilly, L. A. Marconi, D. Olson, R. Tizard, R. Cate, D. Lo. 1995. Expression of RelB is required for the development of thymic medulla and dendritic cells. Nature 373:531.[Medline]
26. Weih, F., D. Carrasco, S. K. Durham, D. S. Barton, C. A. Rizzo, R. P. Ryseck, S. A. Lira, R. Bravo. 1995. Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-B/Rel family. Cell 80:331.[Medline]
27. Sekhar, M., H. Kotani, S. Doren, R. Agarwal, G. McGarrity, C. E. Dunbar. 1996. Retroviral transduction of CD34-enriched hematopoietic progenitor cells under serum-free conditions. Hum. Gene Ther. 7:33.[Medline]
28. Koeffler, H. P., D. W. Golde. 1978. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science 200:1153.[Abstract/Free Full Text]
29. Koeffler, H. P., M. Bar-Eli, M. C. Territo. 1981. Phorbol ester effect on differentiation of human myeloid leukemia cell lines blocked at different stages of maturation. Cancer Res. 41:919.[Abstract/Free Full Text]
30. Kiss, Z., E. Deli, M. Shoji, H. P. Koeffler, G. R. Pettit, W. R. Vogler, J. F. Kuo. 1987. Differential effects of various protein kinase C activators on protein phosphorylation in human acute myeloblastic leukemia cell line KG-1 and its phorbol ester-resistant subline KG-1a. Cancer Res. 47:1302.