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Originally published online as doi:10.1189/jlb.1003501 on January 23, 2004

Published online before print January 23, 2004
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(Journal of Leukocyte Biology. 2004;75:671-679.)
© 2004 by Society for Leukocyte Biology

Induction of various immune modulatory molecules in CD34+ hematopoietic cells

Oliver Umland*, Holger Heine*, Michaela Miehe{dagger}, Kathleen Marienfeld*, Karl H. Staubach{ddagger} and Artur J. Ulmer*,1

* Department of Immunology and Cell Biology, Research Center Borstel, Germany;
{dagger} Centre for Molecular Neurobiology Hamburg, University Hospital Hamburg, Germany; and
{ddagger} Clinic for Surgery, University Hospital Lübeck, Germany

1Correspondence: Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail: ajulmer@fz-borstel.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipopolysaccharide (LPS) has been shown to induce proliferation of human T-lymphocytes only in the presence of monocytes and CD34+ hematopoietic cells (HCs) from peripheral blood. This finding provided evidence of an active role of CD34+ HCs during inflammation and immunological events. To investigate mechanisms by which CD34+ HCs become activated and exert their immune-modulatory function, we used the human CD34+ acute myeloid leukemia cell line KG-1a and CD34+ bone marrow cells (BMCs). We showed that culture supernatants of LPS-stimulated mononuclear cells (SUPLPS) as well as tumor necrosis factor {alpha} (TNF-{alpha}), but not LPS alone, can activate nuclear factor-{kappa}B in KG-1a cells. By cDNA subtraction and multiplex polymerase chain reaction, we revealed differential expression of cellular inhibitor of apoptosis protein-1, inhibitor of {kappa}B (I{kappa}B)/I{kappa}B{alpha} (MAD-3), and intercellular adhesion molecule-1 (ICAM-1) in SUPLPS-stimulated KG-1a cells and up-regulation of interferon (IFN)-inducible T cell-chemoattractant, interleukin (IL)-8, macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), MIP-1ß, RANTES, CD70, granulocyte macrophage-colony stimulating factor, and IL-1ß in stimulated KG-1a cells and CD34+ BMCs. Although monokine induced by IFN-{gamma}, IFN-inducible protein 10, and IFN-{gamma} were exclusively up-regulated in KG-1a cells, differential expression of monocyte chemoattractant protein-1 (MCP-1), macrophage-derived chemokine, myeloid progenitor inhibitory factor-2, and IL-18 receptor was only detectable in CD34+ BMCs. More importantly, CD34+ BMCs stimulated by TNF-{alpha} also showed enhanced secretion of MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8, and increased ICAM-1 protein expression could be detected in stimulated KG-1a cells and CD34+ BMCs. Furthermore, we revealed that T cell proliferation can be induced by TNF-{alpha}-stimulated KG-1a cells, which is preventable by blocking anti-ICAM-1 monoclonal antibodies. Our results demonstrate that CD34+ HCs have the potential to express a variety of immune-regulatory mediators upon stimulation by inflammatory cytokines including TNF-{alpha}, which may contribute to innate- and adaptive-immune processes.

Key Words: innate-immune modulation • T cell activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. LPS stimulates a variety of cell types including monocytes/macrophages [1 , 2 ] and dendritic cells [3 , 4 ], which respond with the release of proinflammatory mediators such as interleukin-1 (IL-1), IL-6, IL-8, IL-12, and tumor necrosis factor {alpha} (TNF-{alpha}), which exerts a wide range of biological activities on many cell types including endothelial cells [5 ], fibroblasts [6 ], and hematopoietic stem cells (HSCs) [7 8 9 ]. Stimulation of TNF receptors (TNFRs) by its ligands leads to the activation of transcription factors such as nuclear factor (NF)-{kappa}B/Rel, which is known to activate genes important in the regulation of inflammatory processes such as cytokine and chemokine production and up-regulation of adhesion and costimulatory molecules [10 ].

LPS also induces the proliferation of human as well as murine T-lymphocytes [11 12 13 14 15 ]. We have previously demonstrated that activation of human T cells by LPS requires the presence of monocytes and CD34+ hematopoietic cells (HCs) from peripheral blood cells (PBCs) [16 ], demonstrating for the first time a direct contribution of CD34+ PBCs in an inflammatory and/or immunological process. Furthermore, we could show that the human CD34+ acute myeloid leukemia cell line KG-1a can functionally replace CD34+ PBCs during stimulation of T cells by LPS [16 ].

In the last few years, early HSCs/hematopoietic progenitor cells (HPCs) became accessible for transplantation using antibody-based selection techniques for stem-cell markers such as CD34 or CD133 [17 , 18 ]. Most reports available to date mainly focused on the characterization or improvement of mobilization and ex vivo expansion strategies for blood stem cells. Recent reports indicate that CD34+ HPCs have the potential to secrete growth factors, cytokines, and chemokines, providing cross-talk mechanisms that regulate various stages of normal human hematopoiesis [19 ]. Until now, however, little is known about direct immune-regulatory functions of CD34+ HCs during inflammation. To elucidate the mechanism by which CD34+ HCs become activated by LPS, directly or indirectly, and how these CD34+ HCs exert their inflammatory and/or immunological functions during the activation of T cells, we made use of the human CD34+ acute myeloid leukemia cell line KG-1a and CD34+ bone marrow cell (BMC) preparations. Here, we demonstrate that supernatants of LPS-stimulated peripheral blood mononuclear cells (PBMCs; SUPLPS), as well as TNF-{alpha} but not LPS alone, can induce translocation of NF-{kappa}B in KG-1a cells. In addition, we can show that stimulation of CD34+ HCs by inflammatory cytokines including TNF-{alpha} leads to the up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression as well as secretion of a variety of immune-modulatory molecules, which may be involved in the recruitment and activation of T cells. Furthermore, we can demonstrate that TNF-{alpha}-stimulated KG-1a cells are able to induce proliferation among T cells, which in turn can be prevented by blocking antibodies to ICAM-1. Therefore, we assume that direct cell–cell interactions between CD34+ HCs and T cells might be involved in the induction of T cell proliferation by LPS as well as other inflammatory and immunological events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and cell culture
The human CD34+ acute myeloid leukemia line KG-1a (American Type Culture Collection, Manassas, VA, No. CCL-246.1) was maintained in Iscove’s modified Dulbecco’s modified Eagle’s medium supplemented with 1% penicillin/streptomycin solution (Gibco/Invitrogen Corp., Karlsruhe, Germany) and 20% heat-inactivated fetal calf serum (FCS; Linaris, Wertheim-Bettingen, Germany) in 25 cm2 cell-culture flasks obtained from Sarstedt (Nümbrecht, Germany).

Preparation of supernatants from human SUPLPS
Human PBMCs were isolated from heparinized blood by density gradient centrifugation over Biocoll separation solution (Biochrom KG, Berlin, Germany) from adult healthy donors [20 ]. PBMCs were washed twice with RPMI 1640 (Gibco/Invitrogen Corp.), resuspended in a concentration of 1 x 106/ml in RPMI 1640, supplemented with 10% heat-inactivated human serum (HS), obtained from adult healthy donors, and incubated with highly purified LPS (with no Toll-like receptor 2-dependent bioactivity) from Salmonella enterica servar friedenau (500 ng/ml, kindly provided by Dr. Helmut Brade, Research Center Borstel, Germany) in a volume of 20 ml in 25 cm2 cell-culture flasks (Sarstedt). Supernatants were harvested after 16 h or as indicated in Results, aliquoted and stored at −20°C until further use.

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
KG-1a cells were stimulated with medium alone, TNF-{alpha} (10 ng/ml, kindly provided by Dr. Daniela Männel, Regensburg, Germany), or SUPLPS (1:2 diluted) in the presence or absence of the anti-TNF-{alpha} antibody Infliximab (100 ng/ml, Centocor Inc., Malvern, PA) or with different stimuli as indicated in Results.

The preparation of nuclear extracts was performed as described elsewhere [21 ]. The oligonucleotide containing the NF-{kappa}B-binding sequence of the murine immunoglobulin (mIg) {kappa} light-chain enhancer [22 ] was obtained from MWG-Biotech GmbH (Ebersberg, Germany). The oligonucleotide (1.25 pmol) was end-labeled in the presence of {gamma}-[32P]deoxyadenosine 5'-triphosphate using T4-polynucleotidekinase (Boehringer Mannheim, Mannheim, Germany) for 30 min at 37°C. Unincorporated nucleotides were removed using a Nick-S-column (Pharmacia, Freiburg, Germany). Labeled oligonucleotides (7.5 fmol) were used in the DNA-binding reaction containing 2 µg crude nuclear extract. The binding reaction also contained 1 µg poly/dI x dC, 1 µg poly/dA x dT, 4% Ficoll, 1 mM dithiothreitol, 2 mM MgCl2, 0.03% Nonidet P-40, and 60 mM KCl. Reactions were incubated for 20 min at 4°C and separated by electrophoresis in 4% polyacrylamide gels containing 0.5 x Tris-boric acid EDTA buffer (45 mM Tris-borate, 1 mM EDTA). Gels were run at 200 V for 1.5 h, sealed, exposed overnight to a phosphorscreen, and analyzed with a PhosphorImager (Molecular Dynamics, Krefeld, Germany).

cDNA subtraction and differential screening
Briefly, KG-1a cells (1x106/ml) were incubated with SUPLPS (1:2 diluted) or RPMI 1640 containing 10% heat-inactivated HS. After 1 h, cells were harvested and washed twice in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4x2H2O, 1.5 mM KH2PO4, pH 7.4), and total RNA was extracted by TRIZOL reagent (Life Technologies/Invitrogen Corp., Karlsruhe, Germany) in accordance with the instructions given by the manufacturer. The integrity of total RNA was analyzed on a 1.5% agarose gel. The mRNA isolation was performed with µMACS mRNA isolation kit (Miltenyi Biotec GmbH, Gladbach, Germany), followed by reverse transcription (RT) for 90 min with 200 U Superscript II (Life Technologies/Invitrogen Corp.). A cDNA synthesis primer (10 µM), delivered within the polymerase chain reaction (PCR)-select cDNA subtraction kit (BD Clonetech, Heidelberg, Germany), primed RT.

cDNA subtraction and differential screening of the subtracted cDNA library was performed by the PCR-select cDNA subtraction kit and the PCR-select differential screening kit (BD Clonetech), respectively, in accordance with the instructions given by the manufacturer. The subtracted cDNA library was screened with [32P]-labeled probes and exposed overnight to a phosphorscreen. A PhosphorImager (Molecular Dynamics) evaluated differential expression. Clones, which were up-regulated more than 2.5-fold, were grown overnight in 30 ml Luria-Bertani cultures at 37°C and 220 rpm. The Nucleobond AX 100 midiprep kit (Macherey-Nagel, Dürren, Germany) prepared plasmid DNA in accordance with the instructions given by the manufacturer and sequenced by MWG-Biotech GmbH.

Isolation of CD34+ HCs from bone marrow
Human BMCs were isolated from the femoral bone of patients suffering necrosis of the head of femoral bone by rinsing the bone marrow with RPMI 1640 obtained from Gibco/Invitrogen Corp. BMCs were passed through nylon net filters (180 µm, Millipore, Eschborn, Germany) and washed twice with RPMI, and finally, BMCs were depleted of erythrocytes by treatment with VitaLyse obtained by Biocarta (Hamburg, Germany) in accordance with the instructions given by the manufacturer.

BMCs were passed through a magnetic cell sorter (MACS) preseparation filter (30 µm), and CD34+ HCs were isolated by positive selection from BMCs using the CD34 progenitor cell isolation kit and LS+ separation columns (Miltenyi Biotec GmbH) as described elsewhere [16 ]. For studying the ICAM-1 expression in CD34+ cells, CD34+ BMCs were cultured as described below. To enhance the purity of CD34+ BMCs for RT-PCR and enzyme-linked immunosorbent assay (ELISA) procedures, MACS-separated CD34+ BMCs were stained with anti-CD34–fluorescein isothiocyanate (FITC; Medac, Wedel, Germany) and anti-CD3–, -CD16–, -CD19 (Dako, Hamburg, Germany)–, -CD14 (Beckman-Coulter, Krefeld, Germany)–, -CD11b–, and -CD56–phycoerythrin (PE) monoclonal antibody (mAb; BD, Heidelberg, Germany) in PBS containing 1% heat-inactivated FCS and were finally sorted by fluorescein-activated cell sorter (FACS; FACSVantage SE, BD) for CD34high/lin– cells. The purity of MACS and FACS was greater than 99% (Fig . 1A 1B 1C 1D ). CD34+ HCs (1x106/ml) were cultured in RPMI 1640, supplemented with 10% heat-inactivated HS in the presence or absence of different reagents or SUPLPS in a volume of 200 µl in 24-well plates (Nunc, Wiesbaden, Germany) or as indicated in Results.



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  		Figure 1. Isolation of CD34+ BMCs by MACS and FACS separation. CD34+ BMCs were isolated from BMCs by MACS separation as described in Materials and Methods, stained with anti-CD34–FITC and anti-CD3–, anti-CD11b–, anti-CD14–, anti-CD16–, anti-CD19–, and anti-CD14–PE, and analyzed by FACS (A and B). FACS further purified presorted CD34+ BMCs for cells expressing CD34high, and PE+ and PE+/CD34+ cells were removed. FACS analyzed sorted CD34+ BMCs (C and D). SSC and FSC, Side- and forward-scatter, respectively.

 
Flow cytometry
KG-1a cells or CD34+ BMCs (1x106/ml) were incubated with RPMI containing 10% heat-inactivated HS, supplemented with or without different amounts of stimuli or SUPLPS (diluted 1:2). After 24 h or as indicated in Results, cells were harvested, washed once, and incubated in azide–PBS (3 mM NaN3, 121.5 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4x2H2O, 1.5 mM KH2PO4, pH 7.4), supplemented with 10% heat-inactivated HS in the presence of anti-CD54 (ICAM-1), anti-CD106 [vascular cell adhesion molecule (VCAM)], anti-CD11a [lymphocyte function-associated antigen-1 (LFA-1)], anti-CD62E [endothelial leukocyte adhesion molecule (ELAM)], or matching IgG1 isotype (all from Chemicon International, Hofheim, Germany). After 30 min, KG-1a cells were washed twice in azide–PBS and incubated for 30 min with goat anti-mouse FITC in azide–PBS containing 10% heat-inactivated HS. ICAM-1 expression on CD34+ cells was analyzed by three-color immunofluorescence using anti-CD34–antigen-presenting cell (APC), anti-CD45–FITC (Medac), and anti-CD54–PE (Chemicon International) or anti-CD45–FITC, IgG1–APC (Medac), and IgG1–PE (BD) as isotype controls. Cells were washed once, resuspended in azide–PBS and an equal volume of paraformaldehyde (3%), and analyzed by flow cytometry using a FACScalibur (BD).

Multiplex RT-PCR
Briefly, KG-1a cells or highly purified (>99%) CD34+ BMCs (1x106/ml) were incubated with RPMI 1640 supplemented with 10% heat-inactivated HS in the presence or absence of TNF-{alpha} (100 ng/ml) or SUPLPS (1:2 diluted). After 16 h, cells were harvested, washed twice with PBS, and lysed by lysis/binding buffer purchased by Dynal (Hamburg, Germany). As a control, PBMCs (1x106/ml) were stimulated in the presence of KG-1a cells (1x105/ml) for 24 h with LPS (500 ng/ml) and lysed as described above. Lysed cells were homogenized at 10,000 g for 2 min at RT using Qiashredder columns (Qiagen, Hilden, Germany). The mRNA preparation was performed with the Dynabeads mRNA direct micro kit purchased by Dynal in accordance with the instructions given by the manufacturer. The mRNA was reverse-transcribed for 50 min with 200 U Superscript II (Life Technologies/Invitrogen Corp.). Resulting cDNA fragments (0.5 µl) were amplified for 33 cycles (hot start for 2 min at 95°C; denaturing at 95°C for 15 s; annealing at 50–58°C for 30 s; elongation at 68°C for 60 s) in an Eppendorf Mastercycler gradient (Eppendorf, Hamburg, Germany) using the AccuPrime Taq DNA polymerase system (Invitrogen) with 0.5 µl AccuPrime Taq and 1 µl primer mix containing 10 µM each primer (Table 1 ) per 20 µl reaction. Amplified products (10 µl) were electrophoresed on a 2% agarose gel, stained by ethidium bromide, and analyzed on a ChemiDoc gel documentation system using Quantity One 4.1.1 software (Bio-Rad, München, Germany).


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  		Table 1. Sequences of Primers Used in RT-Multiplex PCR and/or Quantitative RT-PCR

 
Quantitative RT-PCR
The mRNA isolation and cDNA synthesis for quantitative PCR were conducted as described for multiplex PCR. Quantitative PCR was performed in a LightCycler instrument (Roche Diagnostics, Mannheim, Germany), using 2 µl cDNA (diluted 1:100 for KG-1a cells) and 8 µl PCR master reaction (Fast Start DNA Master SYBR Green I, Roche Diagnostics). For quantification, several dilutions of human SUPLPS cDNA were used as an internal standard, and calculation was performed using LightCycler software version 3.5.3 and the second derivative maximum algorithm. Values were normalized to GAPDH expression, and expression of examined mRNAs in unstimulated cells was set as one. Data are given as x-fold expression.

ELISA
Highly purified (>99%) CD34+ BMCs (1x106/ml) were incubated with RPMI 1640, supplemented with 10% heat-inactivated HS in the presence or absence of TNF-{alpha} (100 ng/ml) or LPS (500 ng/ml). After 16 h, the supernatants were collected and stored at −20°C until use. Human MIP-1{alpha}, MIP-1ß, MCP-1 (R&D Systems, Wiesbaden-Nordenstadt, Germany), or human IL-8 ELISA (Biosource, Solingen, Germany), in accordance with the instructions given by the manufacturer, evaluated the secretion of different chemokines by CD34+ BMCs.

T cell proliferation assay
Human PBMCs were isolated from heparinized blood as described above. Lymphocytes were separated from monocytes using a J2-21 M/E elutriator (Beckman Instruments, UK). T cells were purified by magnetically depleting non-T cells using the Pan T cell isolation kit (Miltenyi Biotec GmbH). These T cells consisted of more than 99% CD3+ T cells as determined by FACS analysis. T cells (1x106/ml) were cultured in the presence of 10% {gamma}-irradiated KG-1a cells (50 Gy) in 12-well plates with medium or TNF-{alpha} (100 ng/ml) in the presence or absence of anti-CD81 (Leinco-Technologies, St. Louis, MO), anti-CD54 (ICAM-1), or matching isotype mAb (100 ng/ml, azide-free) obtained by BD. After 7 days, T cell proliferation was determined by staining for the intracellular proliferation marker Ki-67. Briefly, cells were harvested, stained by anti-CD3–APC, fixed, and permeablized using the Dako IntraStain fixation and permeabilization kit (Dako) and were finally stained for Ki-67 expression by MIB-1 antibody (Dako) conjugated by AlexaFluor488 using the AlexaFluor488 labeling kit (MoBiTec, Göttingen, Germany). Ki-67 expression in CD3+ T cells was determined by FACS analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of NF-{kappa}B translocation in KG-1a cells by cytokines
To test the direct stimulation of CD34+ HCs by bacterial LPS or by cytokines, which are derived from LPS-stimulated monocytes, we first used the human CD34+ acute myeloid leukemia cell line KG-1a as a relevant model. In a first set of experiments, KG-1a cells were stimulated with LPS, TNF-{alpha}, IL-1ß, IL-6, or GM-CSF. The results indicate that LPS alone does not stimulate the translocation of NF-{kappa}B in KG-1a cells, and among the cytokines tested, only TNF-{alpha} is able to activate KG-1a cells (Fig. 2 ).



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  		Figure 2. NF-{kappa}B translocation in KG-1a cells after stimulation with various cytokines or LPS. KG-1a cells (1x106/ml) were incubated with medium (M), TNF-{alpha} (10 ng/ml), IL-1ß (100 U/ml), IL-6 (10 ng/ml), GM-CSF (500 U/ml), or LPS (500 ng/ml). After 1 h of incubation, nuclear extracts were prepared and tested for translocation of NF-{kappa}B by EMSA.

 
In a next set of experiments, we investigated the stimulatory capacity of culture supernatants from human SUPLPS on KG-1a cells and whether TNF-{alpha} is the main or only stimulatory activity in these supernatants. Therefore, we stimulated KG-1a cells with SUPLPS in the presence of an anti-TNF-{alpha} mAb (Infliximab). We found that a substantial amount of the stimulatory activity of SUPLPS is neutralized by this anti-TNF-{alpha} mAb (Fig. 3 ). In summary, our experiments show that SUPLPS is able to induce the translocation of NF-{kappa}B in KG-1a cells, and TNF-{alpha} is the main, but not only, stimulatory constituent in SUPLPS.



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  		Figure 3. LPS-induced release of stimulatory activity by PBMCs acting on KG-1a cells (1x106/ml), which were stimulated by TNF-{alpha} (10 ng/ml) or SUPLPS (50%) in the presence or absence of an anti-TNF-{alpha} mAb (100 ng/ml). After an incubation time of 30 min, nuclear extracts were prepared and tested for translocation of NF-{kappa}B by EMSA. rh, Recombinant human.

 
Stimulation of transcription of ICAM-1 by TNF-{alpha} in KG-1a cells
NF-{kappa}B is a nuclear factor involved in the initiation of the transcription of a variety of inflammatory genes. To define gene products, which are actually induced in KG-1a cells after stimulation with SUPLPS, we compared the gene-expression pattern of stimulated KG-1a cells versus unstimulated KG-1a cells by cDNA subtraction using a cDNA subtraction method. By this method, we detected 15 differentially expressed mRNAs showing 2.5- to sixfold up-regulation in stimulated KG-1a cells as evaluated by phosphorimaging (data not shown). Sequencing of the corresponding clones resulted in three clones that contained inserts for transcripts regulated by NF-{kappa}B. One of these NF-{kappa}B-regulated transcripts contained a fragment that is coding for ICAM-1, as found by database analysis (data not shown). The other two clones contained fragments coding for cIAP-1, a regulator of apoptosis, and I{kappa}B/MAD3, involved in regulation of NF-{kappa}B, respectively. To confirm the differential expression of ICAM-1, cIAP-1, and I{kappa}B/MAD3, we incubated KG-1a cells for 1 h with medium alone or with SUPLPS, and mRNA expression was quantified by real-time PCR after RT. In comparison with unstimulated KG-1a cells, KG-1a cells stimulated for 1 h with SUPLPS showed a marked increase in ICAM-1 and to a lesser extent, in cIAP-1 and I{kappa}B/MAD3 expression. Quantification of ICAM-1, c-IAP-1, and I{kappa}B/MAD3 revealed a 200-fold increase of ICAM-1, a sevenfold increase of cIAP-1, and a ninefold increase in I{kappa}B/MAD3 mRNA in stimulated KG-1a cells (Table 2 ).


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  		Table 2. Differential Expression of ICAM-1, cIAP-1, and I{kappa} B/MAD3 mRNA in KG-1a Cells Analyzed by Quantitative RT-PCR

 
Stimulation of ICAM-1 in KG-1a cells
To investigate whether stimulation of KG-1a cells with SUPLPS not only resulted in transcription of ICAM-1 but also leads to an increase of ICAM-1 protein expression, KG-1a cells were incubated for 24 h with medium alone, SUPLPS, or SUPLPS plus anti-TNF-{alpha} mAb. The expression of ICAM-1 was analyzed by FACS analysis. The results show that stimulation with SUPLPS resulted in a fivefold increase of ICAM-1 protein expression that could be almost completely prevented by preincubation with a neutralizing antibody to TNF-{alpha} (Fig. 4A ).



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  		Figure 4. TNF-{alpha} induces ICAM-1 expression on KG-1a cells (1x106/ml), which were incubated for 24 h under various conditions. Cells were stained with anti-ICAM-1 mAb followed by goat anti-mouse–FITC mAb, and ICAM-1 expression was analyzed by FACS analysis. Results are expressed in a histogram, and corresponding mean fluorescence intensities (Mean FL) are shown in the tables aside. (A) KG-1a cells were incubated with medium alone (unstim.; no) or with SUPLPS (50%) in the presence or absence of an anti-TNF-{alpha} mAb (Ab; 100 ng/ml). (B) KG-1a cells were incubated with medium alone (unstim.; no), TNF-{alpha} (100 ng/ml), or LPS (500 ng/ml).

 
After we had confirmed that SUPLPS can enhance ICAM-1 protein expression in KG-1a cells and that this is mainly a result of TNF-{alpha}, we checked whether LPS or TNF-{alpha} alone can also affect ICAM-1 expression. We incubated KG-1a cells for 24 h with medium alone, TNF-{alpha}, or LPS and examined the ICAM-1 expression by FACS analysis. In contrast to stimulation with LPS, which alone had no effect, stimulation with TNF-{alpha} led to a 20-fold increase of ICAM-1 expression (Fig. 4B) .

Determination of the dose-response effect of TNF-{alpha} (0.01–1000 ng/ml) on KG-1a cells revealed that incubation with 1 ng/ml TNF-{alpha} was sufficient for a significant up-regulation of the ICAM-1 expression on KG-1a cells, and peak expression of ICAM-1 was reached by a TNF-{alpha} concentration of 100 ng/ml. Time kinetics (0–48 h) showed that ICAM-1 is already expressed on KG-1a cells treated for 3 h with TNF-{alpha} (100 ng/ml). The ICAM-1 expression reached a plateau after 12 h and was stable for at least the next 36 h of incubation (data not shown).

Beside ICAM-1, various additional adhesion molecules are involved in cell–cell interaction during inflammation as well as in homing and retention in the tissues. Therefore, we examined whether the expression of other adhesion molecules is also affected by TNF-{alpha} stimulation. KG-1a cells were stimulated with TNF-{alpha} for 24 h, and the expression of LFA-1 (CD11a), ELAM (CD62-E), VCAM (CD106), and ICAM-1 (CD54) was determined by FACS analysis. ELAM and VCAM were neither present before or after incubation with TNF-{alpha}, and LFA-1 was already strongly expressed on unstimulated KG-1a cells and only slightly increased after TNF-{alpha} stimulation. The enhanced ICAM-1 expression was used as a positive control (data not shown).

Stimulation of ICAM-1 in CD34+ BMCs
To confirm the data, which were obtained with the KG-1a cells, we investigated whether ICAM-1 expression is also up-regulated under inflammatory conditions on native CD34+ HCs. Therefore, we prepared CD34+ BMCs by positive selection for CD34 from BMCs isolated from the femur of adult patients. CD34+ BMCs (1x106 /ml) were incubated with medium alone, TNF-{alpha} (100 ng/ml), or SUPLPS in the presence or absence of an anti-TNF-{alpha} mAb. After 24 h, the ICAM-1 expression was determined by FACS analysis on CD34+ cells gated for expression of CD45low/intermediate. As shown in Figure 5 , up-regulation of ICAM-1 expression could be induced on CD34+ BMCs by SUPLPS and to a lesser extent by TNF-{alpha}, and the SUPLPS-induced up-regulation of ICAM-1 could be nearly completely blocked by anti-TNF-{alpha} mAb.



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  		Figure 5. TNF-{alpha} is responsible for induction of ICAM-1 on CD34+ BMC preparations (1x106/ml), which were cultured with medium, SUPLPS (50%) preincubated for 30 min in the presence or absence of an anti-TNF-{alpha} mAb (100 ng/ml), or TNF-{alpha} (100 ng/ml). After 24 h, CD34+ BMCs were stained with anti-CD34–APC, anti-CD45–FITC, and anti-CD54–PE (ICAM-1) or as a control with matching isotypes (not shown). Results for ICAM-1 expression on CD34+ BMC are shown as dot blots.

 
Stimulation of expression of multiple chemokines and costimulatory molecules in KG-1a cells
As differential expression of adhesion molecules is often associated with differential expression of cytokines and costimulatory molecules, we examined whether KG-1a cells have the capacity to express molecules relevant to T cell attraction and/or stimulation. To test our hypothesis, we incubated KG-1a cells (1x106/ml) for 24 h with SUPLPS or medium alone. As a positive control, we used PBMCs (1x106) containing KG-1a cells (10%) stimulated for 24 h with LPS (1 µg/ml) and evaluated the expression profiles by multiplex RT-PCR. Our multiplex PCR consisted of 11 primer sets, encompassing a total of 26 gene products. All mRNAs tested by these primer sets could be amplified in the positive control (except CD70, which was not detectable in the presence of MPIF-2 mRNA), and only 18 were expressed in KG-1a cells. Eleven of them, namely I-TAC, CD70, IL-8, RANTES, MIP-1{alpha}, MIP-1ß, IFN-{gamma}, IP-10, and MIG, were found to be differentially expressed in SUPLPS-stimulated KG-1a cells (Fig. 6 ). Although differences in IL-1ß and GM-CSF expression were poorly visible using multiplex PCR, differential expression could be confirmed by quantitative PCR (data not shown).



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  		Figure 6. Expression pattern of multiple cytokines, chemokines, and costimulatory molecules in KG-1a cells (1x106/ml), which were incubated with medium or SUPLPS. After 24 h, the mRNA was prepared followed by RT and multiplex PCR products, which were separated and analyzed by an ethidium bromide-stained agarose gel (2%). Lanes 1, 4, 7, 11, 14, 17, 20, 23, 26, 30, and 33, Unstimulated KG-1a cells; lanes 2, 5, 8, 12, 15, 18, 21, 24, 27, 31, and 34, SUPLPS-stimulated KG-1a cells; and lanes 3, 6, 9, 13, 16, 19, 22, 25, 28, 32, and 35, LPS-stimulated (24 h) PBMCs (1x106) containing 10% KG-1a cells (control cells). Lanes 10 and 29, DNA marker (M); the positions of the bands are indicated by arrows.

 
Numerous chemokines and costimulatory molecules are differentially expressed and secreted in CD34+ BMCs stimulated by TNF-{alpha}
To investigate whether native CD34+ HCs express a similar pattern of cytokines, chemokines, and costimulatory molecules, like KG-1a cells, we incubated highly purified (>99%) CD34+ BMCs (1x106/ml) with TNF-{alpha} (100 ng/ml), or medium alone. After 16 h, the mRNA was prepared from CD34+ BMCs, and following RT, the expression pattern was analyzed by multiplex PCR. By this method, we observed the up-regulation of IL-18 receptor (IL-18R), MCP-1, MIP-1ß, IL-8, I-TAC, RANTES, MIP-1{alpha}, CD70, IL-1ß, GM-CSF, MDC, and MPIF-2 in CD34+ BMCs stimulated by TNF-{alpha}. Although the expression of IL-18, VLA-4, IL-12R, and PF-4 was not affected by TNF-{alpha} stimulation, down-regulation of CD30L and TGF-ß has been observed (Fig. 7 ).



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  		Figure 7. Expression pattern of various cytokines, chemokines, and costimulatory molecules in CD34+ BMCs (1x106/ml), which were incubated with TNF-{alpha} or medium alone. After 16 h, the mRNA was prepared followed by RT and multiplex PCR products, which were separated and analyzed by an ethidium bromide-stained gel (2%). Lanes 2, 4, 6, 8, 10, 12, 14, 16, 19, 21, 23, 25, and 27, Unstimulated CD34+ BMCs; lanes 3, 5, 7, 9, 11, 13, 15, 17, 20, 22, 24, 26, and 28, TNF-{alpha}-stimulated CD34+ BMCs. Arrows indicate the positions of the bands. Lanes 1 and 18, DNA standard marker (M).

 
To test whether stimulation of CD34+ BMCs with TNF-{alpha} leads not only to increased mRNA expression but also to enhanced protein synthesis and secretion, we incubated CD34+ BMCs (>99 pure) with TNF-{alpha} (100 ng/ml), LPS (500 ng/ml), or medium alone. After 16 h, culture supernatants were collected, and ELISA determined the concentration of MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8, chemokines known to be involved in T cell attraction and/or activation. Although LPS had no effect on the MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8 expression, increased concentrations of those chemokines were detectable in supernatants of TNF-{alpha}-stimulated CD34+ BMCs (Table 3 ).


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  		Table 3. Differential MCP-1, MIP-1{alpha}, MIP-1ß, and IL-8 Protein Expression by CD34+ BMCs

 
TNF-{alpha} can functionally replace monocytes during the induction of T cell proliferation mediated by CD34+ HCs
It has been shown that native CD34+ HCs can be functionally replaced by KG-1a cells during LPS-induced T cell proliferation. In addition, we could show that TNF-{alpha} is capable of inducing a variety of immune-regulatory mediators including ICAM-1 in KG-1a cells and CD34+ BMCs. Therefore, we examined whether T cell proliferation can be induced in the absence of accessory monocytes by TNF-{alpha} in the presence of KG-1a cells alone. Greater than 99% pure T cells (1x106/ml) were cultured in the presence of {gamma}-irradiated KG-1a cells (10%) with TNF-{alpha} (100 ng/ml) or medium alone. The cultures were harvested on day 7 and stained by anti-Ki-67 and anti-CD3 antibodies, and the T cell proliferation was determined by FACS analysis. Although only a negligible amount CD3/Ki67+ T cells were observed in unstimulated cultures, a small CD3/Ki67+ T cell population (2.5%) could be detected in cultures stimulated by TNF-{alpha} (Fig. 8 ). After we have shown that T cell proliferation can be induced by TNF-{alpha}-stimulated KG-1a cells alone, we next wanted to study which molecules expressed by CD34+ HCs are indispensable for T cell activation.



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  		Figure 8. ICAM-1-mediated cell––cell interactions between KG-1a cells and T cells are crucial for TNF-{alpha}-induced T cell proliferation. T cells (1x106/ml) were cultured in the presence of {gamma}-irradiated KG-1a cells (10%) with medium or TNF-{alpha} (100 ng/ml) in the presence or absence of anti-CD81, anti-ICAM-1, or matching isotype antibody (100 ng/ml). After 7 days, cells were harvested and stained by anti-CD3–APC and anti-Ki-67–Alexa488 antibodies. T cell proliferation was determined by FACS analysis. Values are given as percentage of total T cells.

 
ICAM-1-mediated cell–cell interactions between KG-1a cells and T cells are crucial for T cell proliferation
As direct cell–cell interactions are crucial for T cell activation, and ICAM-1 is an essential molecule in forming and maintaining cell adhesion (i.e., forming of the immunological synapse), we examined whether blockage of direct cell–cell interaction by anti-ICAM-1 antibodies can prevent the initiation of T cell proliferation induced by TNF-{alpha}-stimulated KG-1a cells. Therefore, highly purified T cells (1x106/ml) were cultured in the presence of KG-1a cells (10%) with TNF-{alpha} (100 ng/ml) in the presence or absence of blocking anti-ICAM-1 (100 ng/ml), anti-CD81 (100 ng/ml), or matching isotype mAb (100 ng/ml) or with medium alone. After 7 days, cultured cells were stained by anti-Ki-67 and anti-CD3 mAb, and FACS analyzed proliferation.

Although the isotype mAb and the anti-CD81 mAb had only slight effects on T cell proliferation, incubation with a blocking anti-ICAM-1 mAb reduced the T cell proliferation that was induced by TNF-{alpha}-stimulated KG-1a cells by 86% (Fig. 8) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, we could show that CD34+ HCs are required for LPS-induced activation of human T cells [16 ]. This finding demonstrated for the first time an active role of CD34+ HCs in inflammatory and immunological events. Until now, however, little was known about the mechanisms, first by which CD34+ HCs become activated and second by which they exert their inflammatory and/or immunological effector functions. To investigate these questions, we used the human CD34+ acute myeloid leukemia cell line KG-1a and CD34+ BMCs. We could demonstrate that supernatants of SUPLPS can activate NF-{kappa}B in KG-1a cells. Neutralization experiments with anti-TNF-{alpha} mAb showed that TNF-{alpha} is the main stimulatory activity in SUPLPS, and indeed, we could detect activation of NF-{kappa}B in KG-1a cells by TNF-{alpha} alone. This is consistent with the finding of other groups [23 ]. To identify inflammatory genes that may be activated by SUPLPS, we conducted a cDNA subtraction technique and discovered differentially expressed mRNAs including a regulator of apoptosis (cIAP-1), a modulator of the NF-{kappa}B signal-transduction pathway (I{kappa}B/MAD3), and ICAM-1, an adhesion molecule known to be an important participant in T cell activation [24 25 26 ]. Differential expression of ICAM-1 has been confirmed on the transcriptional as well as on the protein level, indicating that CD34+ HCs are able to perform cell–cell interactions and to present costimulatory signals to T cells under inflammatory conditions, and indeed, our experiments using blocking anti-ICAM-1 antibodies could demonstrate that ICAM-1-mediated cell–cell interactions between KG-1a cells and T cells are relevant for induction of T cell activation and proliferation by TNF-{alpha}-stimulated KG-1a cells.

It has been reported that HSC/HPCs have the potential to express a variety of cytokines, chemokines, and growth factors, providing a cross-talk mechanism necessary for normal hematopoiesis [19 ]. As some of those molecules expressed by HSC/HPCs such as RANTES, MIP-1{alpha}, and MIP-1ß are primarily described to be chemotactic for immune-competent cells including T-lymphocytes [27 , 28 ], we decided to screen KG-1a cells and CD34+ BMCs by multiplex PCR for molecules that trigger chemotaxis or are involved in the activation of T cells. We could demonstrate up-regulation of chemokines such as I-TAC, RANTES, MIP-1{alpha}, and MIP-1ß in KG-1a cells and CD34+ BMCs, all of which are known to act on T cell activation and polarization. It has been reported that high expression of MIP-1{alpha} and MIP-1ß secreted by TNF-{alpha}-stimulated CD34+ BMCs and RANTES promotes attraction of a variety of cell types including activated and memory T cells, monocytes/macrophages, and immature APCs [27 , 28 ] as well as up-regulation of IFN-{gamma} in activated T cells in a chemokine receptor (CCR)5-dependent manner [29 ]. CXC-CCR3 and CCR5 are regarded as T helper cell type 1 (TH1) CCRs found to be expressed on 5–15% of peripheral blood T cells, indicating that CD34+ HCs could play a role in promoting TH1 rather than TH2 responses [30 31 32 33 34 ]. Differential expression of MIP-1{alpha}, MIP-1ß, and IL-1ß, which is known to induce IL-2 and IL-2R expression in TH cells, was measured in both CD34+ cell populations investigated. We therefore assume an essential role for these mediators during regulation of inflammatory processes by CD34+ HCs, at least during activation of T cells by LPS. In addition, we found differential expression of MDC, MPIF-2, TGF-ß, IL-8 and GM-SCF. Although MDC [35 ], like MPIF-2, is considered as a T cell attractant, MPIF-2 is also reported to be an efficient inhibitor of HSC proliferation [36 ], a feature shared by IL-8 [37 ], MIP-1{alpha} [38 ], and IP-10. However, which of those mediators expressed by CD34+ HCs in addition to ICAM-1 are indispensable to support T cell activation and recruitment needs further investigations. In contrast with other groups, we observed no IFN-{alpha} and MCP-4 expression by CD34+ HCs (data not shown) [19 ].

It is surprising that we found differential expression of CD70 in KG-1a cells and CD34+ BMCs. CD70 belongs to the TNFR family and is reported to be expressed only transiently upon activation in T and B but not in dendritic cells [39 ]. CD70 seems to play a role in costimulating already-primed T-lymphocytes. Expression of CD27, the receptor for CD70, is strictly confined to T, B, and natural killer cells [40 41 42 43 ] and becomes up-regulated upon ligation with proper antigen/major histocompatibility complexes on T cells [39 ]. Therefore, we hypothesize that expression of ICAM-1 in conjunction with CD70 is maybe responsible for the activation of peripheral blood T cells by TNF-{alpha}-stimulated KG-1a cells, and indeed, preliminary data indicate that anti-CD70 antibodies can reduce the T cell proliferation induced by TNF-{alpha}-stimulated KG-1a cells by ca. 50% (data not shown).

As even highly purified CD34+ HC preparations may still be heterogeneous and may contain cells other than HSCs and HPCs, we cannot exclude that cells other than HSCs and/or HPCs may be the main source of the immune-regulatory molecules that were detected by us. However, it has been shown that different CD34+ HPC populations are capable of expressing a variety of immune modulatory mediators [19 ]. This suggests that these mediators could at least in part indeed be expressed and/or secreted by CD34+ HSCs and/or HPCs.

In conclusion, we could show that CD34+ HCs can be activated by inflammatory cytokines to express a variety of immune-regulatory mediators that are capable of providing helper activity during inflammation and may contribute to the network of the innate- and adaptive-immune system. Furthermore, we have shown that direct, ICAM-1-mediated cell–cell interactions between KG-1a cells and T cells are important for T cell activation, and we assume that such interactions may play a role during the induction of T cell proliferation by LPS in vivo. However, little or nothing is known about the number and location of CD34+ HCs in secondary lymphoid organs and tissues under normal and/or inflammatory conditions. However, even minute numbers of CD34+ HCs have been shown to exert essential accessory function at least in vitro [16 ]. We assume that these CD34+ HCs may play a role during development of chronic ostitis, which might be a good model to investigate immune-regulatory functions of CD34+ HCs in vivo.


   ACKNOWLEDGEMENTS
 
The authors thank the laboratory of Dr. D. Riethmacher (Centre for Molecular Neurobiology Hamburg, University Hospital Hamburg, Germany) for helping us with cDNA subtraction and differential screening technique, Dr. M. Ernst (Department of Cell Biology, Research Center Borstel, Germany) for helping us with FACS analysis, and C. Schneider, I. Goroncy, and K. Klopfenstein for excellent technical assistance.

Received October 23, 2003; revised December 15, 2003; accepted December 16, 2003.


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 DISCUSSION
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