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STEM CELLS AND DEVELOPMENTVolume 19, Number 5, 2010© Mary Ann Liebert, Inc.DOI: 10.1089/scd.2009.0365

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to differentiate into different tissue cell types such as chondrocytes, osteocytes, and adipocytes. In addition, they can home to damaged, in-fl amed, and malignant tissues and display immunomodulatory properties. Since tissue-derived factors might modulate these properties, we decided to explore the impact of prototypic tissue-derived infl ammatory cytok-ines such as TNF-α and IFN-γ on immunomodulatory MSCs functions. To this end, we used primary bone marrow and cord blood-derived MSCs as well as an immortalized MSC line (V54/2) as model systems. We demonstrate that under unstimulated conditions, V54/2 cells constitutively express low levels of indoleamine 2,3-dioxygenase (IDO), exert an immunosuppressive effect on activated T-lymphocyte proliferation, secrete a distinct set of cytokines, and express a wide range of chemokine receptors. Upon stimulation, the proinfl am-matory cytokines IFN-γ and TNF-α did not inhibit suppression of T-cell proliferation, although IDO expression was up-regulated by IFN-γ. In contrast, TNF-α but not IFN-γ amplifi ed the cytokine production of V54/2 and primary MSCs. Interestingly, IFN-γ was superior to TNF-α in up-regulating expression of chemokine receptors and migration of the V54/2 cell line, while TNF-α was the predominant regulator of migration in primary MSCs. Altogether, our data show that properties of MSCs depend on local environmental factors. In particular, we have shown that IFN-γ and TNF-α differentially regulate cytokine expression and migration of MSCs.

Introduction

Mesenchymal stem cells (MSCs) are multipotent pro-genitor cells [ 1 ] that can potentially differentiate into

a variety of cell types, including osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of vis-ceral mesoderm [ 2–5 ]. In vitro, they grow as adherent fi bro-blast-like cells that typically express CD29, CD73, CD90, and CD105 on their surface but lack expression of hematopoietic markers like CD34, CD45, CD14, and CD11 [ 6 ]. Classically, MSCs are raised out of bone marrow-derived mononuclear cell fractions, more recently they can also be grown out of other tissues such as salivary gland, placenta, muscle, adi-pose tissue, connective tissue, umbilical cord blood, and peripheral blood [ 7–10 ].

Recent studies demonstrated that MSCs are capable of supporting hematopoiesis and of regulating immune responses [ 2 ]. In addition, MSCs are thought to play an im-portant role in tissue repair and wound healing and thus are currently being evaluated in various preclinical and

clinical studies [ 11 , 12 ]. Interestingly, MSCs have the ability to accumulate at sites of tissue damage, infl ammation, and cancer. Although in the peripheral blood MSCs are present at very low numbers [ 2 ], they seem to reach specifi c target tissues through the general circulation. The exact molec-ular mechanisms of MSCs traffi cking are still poorly un-derstood. Homing of MSCs has been reported both in vivo and in vitro, and it is believed to rely on adhesion molecules and chemokine receptors via multistep cascade, consisting of a rolling process followed by fi rm adhesion and transend-othelial migration into target tissues [ 13–15 ]. Upon arrival in the target tissue, MSCs interact with various tissue-resident hematopoietic and nonhematopoietic cell types. During this interaction, MSCs are exposed to local tissue-derived in-fl ammatory mediators and immunological signals.

Recent evidence suggests a role for MSCs in immuno-regulation by direct interaction with a variety of myeloid and lymphocytic leukocyte subsets [ 16 ]. In these settings, MSCs mostly have been defi ned as immunosuppressive,

Interferon-γ and Tumor Necrosis Factor-α Differentially Affect Cytokine Expression and Migration Properties

of Mesenchymal Stem Cells

Hatim Hemeda , 1 Mark Jakob , 1,2 Anna-Kristin Ludwig , 3 Bernd Giebel , 3 Stephan Lang , 1 and Sven Brandau 1

1 Department of Otorhinolaryngology, Head and Neck Surgery , University Hospital Essen, Essen, Germany. 2 Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Bonn, Bonn, Germany. 3 Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany.

HEMEDA ET AL. 694

Flow cytometry

For cell-surface marker immunophenotyping, cells were analyzed by direct or indirect immunofl uorescence using specifi c monoclonal antibodies listed in Table 1 . To detect surface antigens, cells were detached with Accutase solution (PAA), washed once with PBS containing 15 mM sodium azide (PBS-azide), and incubated (30 min at 4°C) in Na-azide-PBS containing 3% human serum with the respective anti-body at a concentration previously established by titration. For indirect immunofl uorescence assays, cells were washed and incubated with secondary antibody (α-mouse IgM PE; BD Pharmingen, San Diego, CA). Background fl uorescence was subtracted after analyzing cells stained with the rel-evant isotype control. In each case, 10,000 events were ac-quired by fl ow cytometry using the FACSCalibur cytometer (BD Biosciences, San Jose, CA) and the collected data were analyzed with WinMDI 2.8 software.

T-cell proliferation assay

Proliferation assay was performed in fl at-bottomed 96-well plates (Greiner bio-one) in a total volume of 0.2 mL growth medium. CD4 + T cells were purifi ed from periph-eral mononuclear cells from a healthy volunteer donor using CD4 isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Purifi ed CD4 + T cells (10 5 cells) were prelabeled with 5 μM carboxyfl uorescein diacetate succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) and stimulated with 5% allogeneic monocytes, 0.1 μg/mL anti-CD3 mAb (Beckman Coulter), and 0.05 μg/mL anti-CD28 mAb (Beckman Coulter) in the presence or absence of V54/2 cells at dif-ferent ratios. In order to examine the role of proinfl amma-tory mediators on the suppressive activity of MSCs, T-cell proliferation in the presence of IFN-γ or TNF-α pre-stimu-lated V54/2 cells was assessed. Additionally, to determine whether soluble factors released by MSCs are suffi cient to achieve immunosuppression, we supplemented lym-phocyte cultures with conditioned medium derived from V54/2 culture. Plates were incubated for 5 days at 37°C in a humidifi ed atmosphere with 5% CO 2 . Cells were then col-lected, and the proliferation status was analyzed by fl ow cytometry. Events were acquired on a FACSCalibur cytom-eter and the collected data were analyzed with WinMDI 2.8 software.

Cytokine treatment of MSCs and MSC line cells

V54/2 cell line cultures were seeded onto 12-well plates (Greiner bio-one) and cultured in RPMI 1640 with 10% heat-inactivated, FCS, 1% glutamine, and 1% peni-cillin–streptomycin. Confl uent cultures were stimulated with 50 ng/mL SDF-1α (Peprotech), 1,000 U/mL TNF-α (Peprotech), 1,000 U/mL IFN-γ (Peprotech), 100 ng/mL LPS (kindly provided by Prof. Dr. K Brandenburg, Division of Biophysics, Research Center Borstel, Borstel, Germany), 1 μM Poly(I:C) (Sigma-Aldrich, St. Louis, MO), 100 ng/mL IL-1β (Immunotools), or 100 ng/mL MIP-3β (Immunotools) or exposed to a combination of either IFN-γ/TNF-α or IFN-γ/IL-1β. Additionally, bone marrow- and cord blood-derived MSCs were seeded onto 12-well plates and con-fl uent cultures were stimulated with 1,000 U/mL IFN-γ and 1,000 U/mL TNF-α. In each case, RNA or supernatants

down-regulating functions of dendritic cells, T cells, NK cells, and others. IDO activity appears to be a major mechanism of immunosuppression, but other mechanisms may also be involved [ 17–23 ]. The immunobiology of MSCs clearly focused on the immunosuppressive activity and the mecha-nisms involved. It is less clear whether MSCs display other “immune-like” functions and the response of MSCs to typical immunological signals is poorly defi ned. Thus, we wondered whether MSCs could be regarded as “immune-like” progen-itor cells that respond to classical immunological signals and share many functions with myeloid and lymphocytic subsets.

To investigate this in more detail, we exposed both pri-mary MSCs as well as a MSC line to prototypic infl amma-tory mediators and assessed the immunological response of these cells. We also included a MSC line into our study because these cells exhibit reproducible features in immu-nologic assays, are readily and consistently available, and still share many cell biological properties with primary cells. We found that MSCs exhibited many myeloid leu-kocyte features and were highly responsive to classical infl ammatory cytokines. Among those cytokines, IFN-γ and TNF-α differentially regulate specifi c immunological functions of MSCs. Thus, our data suggest a potential role for MSCs as immunoregulatory cells and show that infl am-matory tissue factors differentially regulate specifi c MSCs functions.

Materials and Methods

Isolation of primary human mesenchymal stem cells

Human umbilical cord blood (CB) and bone marrow (BM) were obtained from unrelated donors after informed consent according to the Declaration of Helsinki. Primary human MSCs were generated from mononuclear cells obtained by Ficoll (Biocoll Separating Solution, Biochrom AG, Berlin, Germany) density gradient centrifugation of bone marrow and umbilical cord blood. The mononu-clear cells were cultured in tissue culture 6-well plates at a density of 2 × 10 6 cells per well in α-MEM (Promocell, Heidelberg, Germany) with 10% fetal bovine serum (FBS) (PAN, Aidenbach, Germany), 1% glutamine, and 1% penicillin–streptomycin (PAA, CÖlbe, Germany) for 24 h. After 24 h of incubation, nonadherent cells were removed by medium ex-change. After confl uence had been reached, usually within 14 days, cells were continuously passaged after treatment with 0.25% trypsin. Adherent cells showed fi broblast-like morphology, and were able to differentiate along the adipo-genic, chondrogenic, and osteogenic pathways.

Peripheral blood-derived human mesenchymal stem cell line V54/2

V54/2 is a human MSC line originally generated from MSCs of the peripheral blood. V54/2 has been immortal-ized by SV40 large T antigen [ 24 ]. Cells were generated by Dr. Ralf Huss (Roche, Penzberg, Germany) and were kindly provided by Dr. Peter Nelson (Medical Policlinic, University of Munich, Munich, Germany). V54/2 cells were grown in RPMI 1640 (PAA) with 10% heat-inactivated, fetal calf serum (FCS; Biochrom, AG), 1% glutamine, and 1% penicillin–streptomycin.

MODULATION OF MSC IMMUNOBIOLOGY 695

Adhesion assays

Cell adhesion assays were performed in 96-well plates that were coated with 20 ng/mL Collagen VI (Sigma-Aldrich, St. Louis, MO) at 4°C overnight. Wells were washed 3 times with PBS buffer. Then, a total of 1–10 × 10 4 V54/2 cells were seeded on either precoated or uncoated wells and allowed to adhere for 2.5 h at 37°C. The non-adherent cells were removed by PBS washing, and the adherent cells were resuspended in 100 μL fresh medium containing 20% MTT (Sigma-Aldrich) (5 mg/mL). Cells were incubated at 37°C for 3 h, and then 100 μL of dim-ethyl sulfoxide (DMSO, Roth) was added to each well, and mixed by shaking at room temperature for 10 min. The absorbance was measured at 630 nm using a Synergy HT Multi-Mode Microplate reader (BioTek Instruments, Bad Friedrichshall, Germany). For each set of experi-mental conditions, a minimum of 4 measurements were performed.

RNA isolation and polymerase chain reaction

Total RNA was purifi ed from the MSCs and MSC line cul-tures using NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. Approximately 0.8 μg of total RNA was reverse-transcribed using SuperScript II RNase H − Reverse Transcriptase kit (Invitrogen, Carlsbad, CA), with Oligo dT at 42°C for 50 min in a 20 μL reaction and quantifi ed using Quant-iT RiboGreen RNA kit (Molecular Probes, Eugene, OR). Primers for gene expression analysis of indoleamine 2,3-dioxygenase (IDO) or chemokine receptors (listed in Table 2 ) were identifi ed using OligoExplorer 1.2 software or based on previously published sequence reports in the literature. Each PCR was performed in a 25 μL solution containing 1 μL cDNA as a template, 10× PCR buffer, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 1 μM of sense primer, 1 μM of antisense primer, and 0.5 U Taq DNA polymerase (Invitrogen). PCR amplifi cation con-ditions were: initial incubation 15 min at 95°C, followed by 35–40 cycles of denaturation for 60 s at 94°C, annealing for 45 s at 60°C, extension for 45 s at 72°C, and terminating with 15 min at 72°C. Amplifi cation was performed in a thermocycler (Bio-Rad, Munich, Germany) and amplifi ed DNA fragments were separated on a 2% agarose gel and photographed after ethidium bromide staining.

Semiquantitative RT-PCR analysis of indoleamine 2,3-dioxygenase and chemokine receptor gene expression

Semiquantitative RT-PCR was performed on diluted cDNA samples from both primary cell isolate and immortal-ized MSC line using DyNAmo Capillary SYBR Green qPCR kit (Biocat, Heidelberg, Germany) and Roche LightCycler instrument (Roche, Mannheim, Germany). The relative ex-pression level of the housekeeping gene β-actin was used to normalize chemokine receptor expression in each sample. PCR conditions for all chemokine receptors were: hot start enzyme activation for 10 min at 95°C, 40–50 cycles of dena-turation for 10 s at 95°C and annealing of receptor-specifi c oligonucleotides for 20 s, and fi nally extension 72°C for 15 s. Oligonucleotides used for PCR are given in Table 2 .

were collected after 24 h incubation and stored at −80°C until use. In order to investigate the effect of different proinfl ammatory mediators on the function of the MSC line, V54/2 cells were pre-stimulated with 1,000 U/mL TNF-α, 1,000 U/mL IFN-γ, 20 ng/mL MIF (Macrophage Migration Inhibitory Factor, R&D Systems), and 100 ng/mL MIP-3β for 4 h. After extensive washing with PBS, cells were exposed to 100 ng/mL LPS for 24 h and supernatants were collected and stored at −80°C until use. IL-6 and IL-8 cytokines were quantitated using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) following the instructions of the manufacturer.

Quantifi cation of cytokines in culture supernatants

For cytokine quantifi cation, cells recovered from cultures were washed twice with PBS and incubated for 24 h in fresh medium in the presence or absence of different cytokines as described above. Supernatants were collected by centrifuga-tion and cytokines IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ were quantitated using commercially available multiplex bead-based sandwich immunoassay kit (Human 8-plex; Bio-Rad Laboratories, Munich, Germany) using a Luminex 100 plate reader (Bio-Rad Life Sciences, Munich, Germany). Assays were performed following the manufac-turer’s instructions and data were analyzed using the Bio-Plex Manager software version 4.1.1 (Bio-Rad Laboratories).

T able 1. A ntibodies U sed in T his S tudy , w ith D etails of T heir S ources and M ain A ntigenic C haracteristics

Clone (label) Antigen Source

25.3.1 CD11a Beckman CoulterM1/70.15 CD11b AbcamB-1y6 (PE) CD11c PharmingenM5E2 (PE) CD14 Pharmingen7E4 CD18 Beckman CoulterHD37 (PE) CD19 DAKOMAR4 (PE) CD29 PharmingenWM59 (PE) CD31 Pharmingen581-Class3 (FITC) CD34 Caltag labmAb89 (RPE) CD40 Immunotech515 (PE) CD44 PharmingenHI100 (RPE) CD45 Pharmingen9F10 (PE) CD49d PharmingenHA58 (RPE) CD54 PharmingenB159 (PE) CD56 DAKOLT-TD180 (FITC) CD62L Immuno ToolsM-A712 (FITC) CD71 PharmingenAD2 (PE) CD73 Pharmingen5E10 (FITC) CD90 Pharmingen166707 (FITC) CD105 R&D systems51-10C9 (FITC) CD106 PharmingenYB5.B8 (RPE) CD117 Pharmingen16803 (FITC) CD120a R&D systemsC40-1457 (RPE) CD271 Pharmingen89106 (PE) VEGFR2 R&D systemsSTRO-1 STRO-1 R&D systemsW6/32 (RPE) MHC I DakoI3-RD1 (PE) HLA-DR Beckman Coulter

HEMEDA ET AL. 696

multiple comparison test, ANOVA, with P < 0.05 considered signifi cant.

Results

Phenotypic characterization of MSCs

In culture, V54/2 cells grow as a population of adher-ent cells with a typical fi broblast-like morphology. To test whether V54/2 cells express surface markers characteristic for MSCs, these cells were subjected to a fl ow cytometric analysis as indicated in Table 1 . Results showed that periph-eral blood-derived V54/2 MSCs express a wide range of extracellular matrix proteins, cytokines, and growth factor receptors. As shown in Figure 1 , the cells revealed posi-tive staining for CD71 (transferrin receptor), CD73 (SH4), CD90, CD105 (endoglin), CD117 (c-kit), CD120a (TNFR1; TNF receptor-1), CD271 (LNGFR; low-affi nity nerve growth factor receptor), VEGFR (vascular endothelial growth factor recep-tor), and MHC class I, albeit at low level for some antigens. In addition, V54/2 MSCs lack the expression of hematopoi-etic markers CD14, CD34, and CD45 as well as CD19, CD40 (TNFR; TNF receptor family), STRO-1, and HLA-DR (MHC class II). The addition of IFN-γ to the cultures enhanced the expression of MHC class I and triggers the expression of MHC class II, whereas TNF-α increased the expression of VEGFR (data not shown).

Inhibition of T-cell proliferation is not altered by proinfl ammatory cytokines

Inhibition of T-cell proliferation in vitro is a classical func-tion of MSCs [ 17–21 ]. We tested the effect of the V54/2 cell line

Chemotaxis assay

To study the MSCs migration, V54/2 cells were incubated for 6 h in RPMI 1640 basal medium in the presence of 0.1% FCS (assay medium). Cells were labeled with 10 μM Calcein AM (BD Bioscience, San Jose, CA) for 1 h at 37°C and 5% CO 2 . After labeling, cells were detached from the cell culture plate using Accutase solution, suspended in assay medium, and seeded on a BD Falcon FluoroBlok 24-multiwell insert (5 × 10 5 cells in 0.35 mL assay medium per top chamber). The bottom chambers contained 0.8 mL of assay medium in the presence or absence of different chemokines at concentra-tions based on previously published reports in the literature. To determine whether proinfl ammatory mediators enhanced the migration potential of MSCs, V54/2 cells were pretreated with IFN-γ, TNF-α, or both in combination as indicated above and afterwards allowed to migrate. Cells were incu-bated in the FluoroBlok multiwell insert system for 3 h at 37°C in a humidifi ed atmosphere of 5% CO 2 . Fluorescence of cells that passed through the BD FluoroBlok membrane was acquired from the bottom side of the multiwell insert system at excitation/emission wavelengths of 485/520 nm using a Synergy HT Multi-Mode Microplate reader. Additionally, bone marrow primary MSCs were pretreated or not with IFN-γ and TNF-α and allowed to migrate. Migration assay was terminated after 4 h and migrated cells were counted manually under high-power magnifi cation.

Statistical analysis

Statistical analysis was performed using Sigmaplot 11 statistics software (Erkrath, Germany). Multiple treatment groups were compared to a single control using Dunnett’s

T able 2. P rimer S ets U sed in T his S tudy

Target

Primer sequence Product

Sense Antisense size (bp) T m °

CXCR1 CGCCAGGCTTACCATCCA CAAACAGCGGCACGATGA 127 62CXCR2 GAGGTGTCCTACAGGTGAAAAGC GGGGGCAGGGTAGAGCTGTA 151 62CXCR3 TGGCCGAGAAAGCAGGG AGGCGCAAGAGCAGCATC 138 63CXCR4 GGCCGACCTCCTCTTTGTC GGACGATGGCCAGGTAGC 173 62CXCR5 TTGCCTTGCCAGAGATTCTCTT TGAACCAGGCATGCGTTTC 111 62CXCR6 CATCTCTGCTGGTGTTCATCAGA GAACTGCAGGAAGTCTTGATGCT 114 62CXCR7 TCTTCTCCTACGTGGTGGTCTTCC GACAGGCACTGTGTGACATGC 151 63CCR1 ATAGAGAGGGAATGTAATGGTGGCC CCAAGCCCTTCTCTGGTACACTTAGT 150 62CCR2 CTGAGACAAGCCACAAGCTGAAC CCGCTCTCGTTGGTATTTCTGA 121 62CCR3 CCGGTGATCTACGCCTTTGTT CAGCTTCTCACTAGGAAGGAATGG 117 62CCR4 AAAGCAAGCTGCTTCTGGTTG TTGGTGCAAGGCTTGGG 151 60CCR5 ACAAGAAACTCTCCCCGGGT GAACACCAGTGAGTAGAGCGGA 151 60CCR6 CCCACAATGAGCGGGGA CAAATAGCCTGGAGAACTGCCTG 151 63CCR7 ATTTTCCAGGTATGCCTGTGTCA TGATGGAGTACATGATAGGGAGG 151 62CCR8 CACTAAGGTCCCGCTGCCT GGCAATAAAAGACAGCAAGGAG 151 64CCR9 GCATCTGACTGACCCACCAT GCCTGACATTGTTTTTCTCACAGTAG 151 58CCR10 GAGGCCACAGAGCAGGTTTC CCTGGACATCGGCCTTGTA 103 60CX3CR1 CCAGGCCTTCACCATGG CAGGCCAATGGCAAAGATG 151 60XCR1 ACCATGGCTGTGTGGGTAGCC TCCCCAGGGACAGCAGGA 151 63IDO TGTGAACCCAAAAGCATTTTTC AAAGACGCTGCTTTGGCC 151 60β-Actin AGCGGGAAATCGTGCGTG GGGTACATGGTGGTGCCG 307 60

T m ° indicates melting temperature in degrees Celsius.

MODULATION OF MSC IMMUNOBIOLOGY 697

gene expression. On the basis of these fi ndings, we asked whether infl ammatory mediators could affect the immu-nomodulatory functions of MSCs. To this end, we exposed MSCs to the classical cytokines IFN-γ and TNF-α and tested the effect of cytokine stimulation on T-cell inhibitory activity. We found that IFN-γ and TNF-α stimulation did not show any effect on the inhibitory function of MSCs ( Fig. 2C ).

MSCs produce a distinct set of cytokines under basal culture conditions

It is the aim of this study to investigate central aspects of immunological activity and responsiveness of MSCs. Therefore, cytokine production by MSCs was analyzed using Bio-Plex technology. In the absence of stimulation, low levels of IL-10, IL-4, and IL-2 were detected. MSCs produced

cells on T-cell proliferation in CFSE dilution assays. As shown in Figure 2A , there was a signifi cant dose-dependent reduc-tion in T-lymphocyte proliferation in the presence of MSCs. Signifi cant inhibition of T-cell proliferation was observed even at a 20:1 ratio of T cells to MSCs and almost a complete inhibi-tion was achieved in the presence of MSCs at the ratio of 1:1 (T cell:V54/2). As induction of IDO activity has recently been described as a major T-cell inhibitory mechanism of MSCs, we investigated the expression of IDO by V54/2 cells. RT-PCR analysis demonstrated that MSCs constitutively expressed low levels of IDO mRNA ( Fig. 2B ). In order to investigate whether MSCs can retain their immunosuppressive proper-ties under proinfl ammatory conditions, the effect of IFN-γ and TNF-α on IDO expression was examined. As shown in Figure 2B , IFN-γ signifi cantly induced the expression of IDO mRNA although TNF-α did not show any effect on the IDO

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FIG. 1. Immunophenotyping of mesenchymal stem cells (MSCs). V54/2 cells were subjected to fl ow cytometry with pri-mary antibodies specifi c for selected surface antigens. Data are depicted as histograms with shaded areas representing isotype control staining.

HEMEDA ET AL. 698

Modulation of MSCs cytokine expression by immunological signals

Next, we investigated the effect of immunological signals on the cytokine production of MSCs; V54/2 cells were stim-ulated with IFN-γ, TNF-α, LPS, IL-1β, or MIP-3β for 24 h.

intermediate levels (from 0.05 to 0.25 ng/mL) of GM-CSF, TNF-α, and IFN-γ and particularly high levels (from 3 to 20 ng/mL) of IL-6 and IL-8 ( Fig. 3A ). Thus, MSCs constitutively produce a distinct set of mostly proinfl ammatory cytokines. Interestingly, MSCs also secreted substantial amounts of IFN-γ, typically expressed by T cells and NK cells ( Fig. 3A ).

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**

FIG. 2. Mesenchymal stem cell (MSC) line V54/2 cells inhibit activated T-lymphocyte proliferation and express indoleam-ine 2,3-dioxygenase (IDO). ( A ) CD4 + T cells were activated with 5% allogeneic monocytes, anti-CD3, and anti-CD28 and incubated in the presence or absence of MSCs in graded ratios. Proliferation of responder cells was analyzed by the incor-poration of CFSE using fl ow cytometry. ( B ) IFN-γ but not TNF-α signifi cantly induces IDO mRNA expression in MSC line V54/2. The 24 h cultures of unstimulated or IFN-γ- or TNF-α-stimulated MSCs were lysed and assayed by semiquantita-tive RT-PCR for IDO expression. ( C ) Infl ammatory mediators do not prevent MSCs suppression of T-lymphocyte prolifer-ation. V54/2 were pre-stimulated with 1,000 U/mL IFN-γ or TNF-α for 24 h and incubated with T cells for 5 days. Data are expressed as mean ± standard error of at least 3 independent measurements (* P < 0.05) (** P < 0.001).

MODULATION OF MSC IMMUNOBIOLOGY 699

should occur at sites of infl ammation, in which TNF-α acts as a major regulator of the cytokine expression. In order to verify these results with primary MSCs, the effect of IFN-γ and TNF-α on MSCs derived from bone marrow and cord blood was examined. We found that TNF-α was clearly su-perior to IFN-γ in inducing secretion of IL-8 in both types of primary MSCs ( Fig. 3B ). On the basis of these fi ndings, we asked whether proinfl ammatory mediators could also modulate the response of MSCs to activating ligands such

TNF-α broadly amplifi ed cytokine production by MSCs as it induced expression of infl ammatory cytokines IL-6 and IL-8 along with induction of the “lymphocytic” cytokines IL-2 and IFN-γ. Strikingly, TNF-α also up-regulated the expression of the “growth factor/differentiation” cytokine GM-CSF. Interestingly, IFN-γ did not induce GM-CSF, IL-6, and IL-8 and thus only marginally regulates basal MSCs cy-tokine responses ( Fig. 3A ). These results suggest that a sig-nifi cant up-regulation of the cytokine production by MSCs

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FIG. 3. Constitutive cytokine expression by mesenchymal stem cells (MSCs) and the effect of immunological signals on cytokine production. Cytokine production by MSCs in the absence or presence of stimulation was evaluated. For stimula-tion, V54/2 cell line cultures ( A ) and primary MSCs ( B ) were treated with 1,000 U/mL IFN-γ, 1,000 U/mL TNF-α, 100 ng/mL LPS, 100 ng/mL IL-1β, or 100 ng/mL MIP-3β as indicated. Supernatants were collected after 24 h incubation and cytokines IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ were quantitated using commercially available multiplex bead-based sandwich immunoassay kits (Bio-Plex) analysis. High standard curves for each soluble factor were used. ( C ) The effect of infl ammatory mediators on the response of MSC line to LPS. V54/2 cells were pre-stimulated with 1,000 U/mL TNF-α, 1,000 U/mL IFN-γ, 20 ng/mL MIF, and 100 ng/mL MIP-3β for 4 h followed by LPS exposure. IL-6 and IL-8 cytokines in culture supernatants were quantitated by ELISA. Data are expressed as mean ± standard error of 3 independent measurements.

HEMEDA ET AL. 700

examined the effect of proinfl ammatory mediators on the adherence of MSCs; cells were treated with TNF-α, IFN-γ, or MIF and allowed to adhere on Collagen VI as described above. While TNF-α but not IFN-γ reduced adhesion to Collagen VI, adhesion of MSCs was slightly increased in the presence of MIF ( Fig. 5B ). This alteration of adhesion to Collagen VI occurred despite the fact that IFN-γ, TNF-α, and MIF (and also IL-1β) did not modulate surface expression level of adhesion molecules (data not shown).

Regulation of chemokine receptor expression and migration

Migration is of pivotal importance for immune cells to exert their specifi c functions. We determined gene expres-sion levels of chemokine receptors in our MSC line. Our data show that MSCs express a broad panel of receptors for chemokines of all 4 subfamilies. Under basal conditions, in the absence of any immunological signals, V54/2 MSCs were positive for CXCR1 to CXCR7, CCR1, CCR3, CCR5–CCR9, CX3CR1, and XCR1 but not for CCR2, CCR4, and CCR10 ( Fig. 6 ). To test whether chemokine receptor expression in blood-derived MSC line is responsive to immunological signals, 50 ng/mL SDF-1α, 1,000 U/mL TNF-α, 1,000 U/mL IFN-γ, 100 ng/mL LPS, 1 μM Poly(I:C), 100 ng/mL IL-1β, 100 ng/mL MIP-3β, or a combination of either IFN-γ/TNF-α or IFN-γ/IL-1β was added to MSCs cultures for 24 h. RNA was extracted and the expression of selected chemokine recep-tors was determined using qPCR analysis. Among tested

as LPS. To this end, we pre-stimulated MSCs with TNF-α, IFN-γ, MIF, and MIP-3β for 4 h before exposure to LPS. MIF was chosen because it is known to modulate LPS re-sponsiveness of target cells [ 25 ]. However, in the majority of settings infl ammatory mediators did not further induce cytokine responses over levels of LPS alone. Solely, TNF-α further enhanced LPS-induced IL-8, while IFN-γ further enhanced LPS-induced IL-6 ( Fig. 3C ). Taken together, these experiments show that human MSCs are capable of pro-ducing a wide range of functionally divergent cytokines. This production can be further amplifi ed after exposure of MSCs toward TNF-α.

TNF-α and MIF modulate adherence of MSCs

To determine the expression pattern of adhesion mol-ecules, MSCs were probed with monoclonal antibodies specifi c for integrin α4 (CD49d), αL, αM, αX (CD11a, b, c, respectively), β1 (CD29), β2 (CD18), l -selectin (CD62L), H-CAM (CD44), ICAM-1 (CD54), N-CAM (CD56), PECAM-1 (CD31), and V-CAM-1 (CD106). H-CAM, β1-integrin, and α4-integrin showed a high mean fl uorescence, followed by V-CAM-1 ( Fig. 4 ). αL-, αM-, αX-, β2-integrins, N-CAM, PECAM-1, ICAM-1, and l -selectin were not detectable by fl ow cytometry. In order to obtain a functional correlation between adhesion molecules and cell adhesive properties, cell adhesion assays were performed. In 2.5 h adhesion assays, we found that collagen VI supported ~2-fold greater static adhesion compared to controls ( Fig. 5A ). Next, we

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MODULATION OF MSC IMMUNOBIOLOGY 701

TNF-α in combination had a minimal effect on the migra-tory response of V54/2 cells ( Fig. 8B ). Interestingly, in pri-mary MSCs TNF-α but not IFN-γ enhanced the migration potential of MSCs in response to RANTES and MEC ( Fig. 8C ). Taken together, these data show that IFN-γ and TNF-α up-regulate a specifi c set of chemokine receptors in MSCs, while both cytokines differentially regulate migration of primary and cell line MSCs.

Discussion

MSCs are present in many compartments of the body including bone marrow and peripheral blood. Circulating MSCs have been described to enter sites of infl ammation, traumatized, and malignant tissues. This feature has even stimulated investigators to explore the capacity of MSCs as therapeutic cell-based delivery vehicles [ 26 ]. Upon arrival in the target tissue, MSCs receive a multitude of tissue-derived signals and interact with other cell types present in the target tissue. The migration of MSCs to infl ammatory and malignant tissues raises the following questions: (i) How do the cells get to the sites of injury or target tissues? (ii) What are the distinct signals that guide migration of MSCs? (iii) Upon arrival in the target tissue, how do MSCs react in response to infl ammatory mediators? (iv) And addition-ally, what are the functional changes of MSCs under these conditions? To address some of these questions, we used a human blood-derived MSC line model. For our experiments, we mainly used a well-characterized human MSC line that shares many cell biological properties with primary MSCs. In contrast to primary cells, cell line models have the ad-vantage of being easy to handle, represent an unlimited self-replicating cell source, exhibit a relatively high degree of homogeneity, and therefore increase reproducibility of the results. Immunologic key functions such as cytokine ex-pression and migration were also assessed in primary MSCs from bone marrow and cord blood.

In the current study, we tested important immune-rel-evant features of V54/2 cells. First, we performed studies

mediators on V54/2 cell line, TNF-α and IFN-γ were found to be major regulators of chemokine receptor expression. Exposure of MSC line to TNF-α induced gene expression of the chemokine receptors CCR3, CCR4, and CXCR4, whereas treatment with IFN-γ up-regulated the expression of the chemokine receptors CCR1, CCR3, and CXCR4 ( Fig. 7A and data not shown). In a next series of experiments, the effect of IFN-γ and TNF-α was also tested on primary MSCs. Here, TNF-α but not IFN-γ strongly up-regulated the expression of CCR5 and CCR10 in primary MSCs ( Fig. 7B ). To assess the migration potential of MSCs and to ensure that the expressed chemokine receptors were functional, chemokine ligands for chemokine receptors were selected. Here, we examined the ability of MSCs to migrate in response to CCL5/RANTES, CCL17/TARC, chemokine with 6 cysteines (CCL21/6Ckine), CCL28/MEC, CXCL12/SDF-1α, and CXCL13/BCA-1 using Falcon FluoroBlok transwell inserts. As shown in Figure 8A , all chemokines tested except CXCL13/BCA-1 signifi cantly induced MSC line migration compared to control conditions. Finally, we tested whether immunological stimulation of MSCs could enhance their migration. Pre-stimulation with IFN-γ led to a signifi cant increase in the migratory response to CCL5/RANTES, CCL28/MEC, and CXCL12/SDF-1α, whereas TNF-α pre-stimulation enhanced the migration of V54/2 only in response to CCL28/MEC ( Fig. 8B ). IFN-γ and

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HEMEDA ET AL. 702

VEGFR + , MHC class I + , CD14 − , CD19 − , CD34 − , CD40 − , CD45 − , STRO-1 − , and HLA-DR − . The higher expression of CD105 and CD117 in bone marrow-derived MSCs exemplifi es their important role in the interaction with hematopoietic stem cells by infl uencing their homing and differentiation [ 29 ]. Previous reports showed that bone marrow-derived MSCs

focusing on the cell phenotype under basal unstimulated conditions. Until now, no single specifi c marker has been identifi ed for MSCs [ 27 ]. Instead, it is believed that MSCs share features with mesenchymal, epithelial, and endothelial cells [ 28 ]. Cells grown under unstimulated conditions were CD71 + , CD73 + , CD90 + , CD105 + , CD117 + , CD120a + , CD271 + ,

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FIG. 7. Modulation of chemo-kine receptor expression. Relative gene expression of chemokine receptors in response to dif-ferent proinfl ammatory agent. Mesenchymal stem cell (MSC) line ( A ) and primary MSCs ( B ) were incubated for 24 h with: lane 1, medium alone (control); lane 2, IFN-γ; lane 3, TNF-α; lane 4, SDF-1α; lane 5, LPS; lane 6, Poly(I:C); lane 7, IL-1β; lane 8, MIP-3β; lane 9, IFN-γ/TNF-α; lane 10, IFN-γ/IL-1β as indicated. RNA was isolated and subjected to RT-PCR analysis. mRNA ex-pression levels for each target gene were calculated normalized to β-actin. Expression in control medium was set as one.

MODULATION OF MSC IMMUNOBIOLOGY 703

previously [ 38–41 ]. CCL28/MEC (mucosae-associated epithelial chemokine) is a recently identifi ed CC chemokine that is abundantly expressed on mucosal epithelial surfaces in several mucosal tissues, including salivary gland, bronchi, colon, and mammary gland [ 42 , 43 ]. The cognate receptors of CCL28, CCR3, and CCR10 are mainly expressed on T lymphocytes, B lymphocytes, and eosinophils. Therefore, CCL28 and their receptors may help unifying the “common mucosal immune system” and play a key role in the segrega-tion and the compartmentalization of the mucosal immune system through recruitment of immune cells to specifi c loca-tions. Strikingly, our data demonstrated that a high migra-tion potential of blood-derived MSC line toward CCL28/MEC is even induced in the presence of proinfl ammatory mediators. These data point to a major role of this chemokine in MSCs homing to mucosal environment. Interestingly, a recent study demonstrated that human CCL28 RNA is also highly expressed by the epithelioid tumor cell line, HCT 116, derived from colon carcinoma, as well as by colorectal ad-enocarcinoma cell lines, DLD-1 [ 43 ] suggesting a potential involvement of CCR3 in migration of MSCs into malignant tissues and provide new aspects in the mechanisms involved in MSCs homing.

It was a major aim of this study to investigate the response of MSCs to immune mediators that are induced or up-regulated in tissues under infl ammatory or malignant conditions. Among the molecules tested, clearly IFN-γ and TNF-α had the most prominent effect on MSCs immunobi-ology. IFN-γ enhanced the expression of MHC class I and triggered the expression of MHC class II. Only IFN-γ but not TNF-α induced expression of IDO, a key molecule impli-cated in MSCs T-cell suppressive activity.

In infl amed target tissue, MSCs will be exposed to a vari-ety of tissue-derived immunomodulatory molecules. In our study, infl ammatory environment was achieved by stimu-lating MSCs mainly with exogenous IFN-γ and TNF-α. We observed that TNF-α but not IFN-γ amplifi ed the cytokine response of MSCs and reduced their adherence to extracel-lular matrix. In contrast, IFN-γ was identifi ed as a major inducer of chemokine receptor expression and migration in the MSC line model, while TNF-α was superior in primary MSCs from bone marrow and cord blood. These data suggest that MSCs functions will be modifi ed and adjusted accord-ing to the specifi c cytokine and chemokine milieu that these cells encounter upon arrival in the target tissue. Croitoru-Lamoury investigated the regulatory effect of IFN-γ and TNF-α on the expression of chemokines and their receptors in bone marrow-derived MSCs at both mRNA and protein levels [ 44 ]. Ponte demonstrated a dramatic increase in bone marrow-derived MSCs sensitivity to RANTES, MDC, and SDF-1α after IFN-γ stimulation and showed that the promi-gratory activity of SDF-1α is increased in TNF-α-primed cells [ 45 ]. We found that migration of cord blood- and bone marrow-derived MSCs was primarily regulated by TNF-α, while IFN-γ was the predominant regulator in the periph-eral blood-derived MSC line.

Previous studies have defi ned MSCs as migratory mes-enchymal cells that can be recruited or directed to sites of infl ammation and cancer. This has led to the concept of MSCs as cell-based delivery vehicles [ 26 , 46–48 ]. Upon arrival in the target tissue, MSCs will be exposed to various tissue-derived local infl ammatory mediators. Today little is known

are highly immunosuppressive [ 17–21 ]. The mechanisms by which MSCs exert immunosuppressive properties is not fully elucidated. Bone marrow-derived MSCs execute their immunomodulatory properties through either cell contact or the production of soluble factors. They can dramatically down-regulate the response of memory T cells [ 20 ] and are able to inhibit the proliferation not only of T cells but also NK cells and B lymphocytes [ 22 ]. Bone marrow-derived MSCs also interfere with DC differentiation, maturation, and function [ 23 ]. In the present study, we show that also cells of a blood-derived MSC line strongly suppress the proliferation of activated lymphocytes in vitro. The mecha-nisms through which MSCs suppress alloresponses are still poorly understood. Nonetheless, it is generally accepted that PGE 2 , immunosuppressive cytokines, and enzymes regulat-ing amino acid metabolism play a major role [ 30 ]. Therefore, expression of IDO, a rate-limiting enzyme, involved in the catabolism of amino acid l -tryptophan into kynurenine, has been documented as a key player in the suppression of T-cell activity [ 31 , 32 ]. Here, we demonstrated that blood-derived MSCs express IDO constitutively, which could be further induced by IFN-γ but not by TNF-α. Interestingly, IFN-γ or TNF-α did not ablate MSCs immunosuppressive activity in accordance with previous reports [ 22 , 33 , 34 ], which is essen-tial to note as MSCs may be required for use in regenerative therapy under infl ammatory conditions. Recent studies on murine bone marrow-derived MSCs reported that the addi-tion of a specifi c IDO inhibitor, the tryptophan antagonist 1-methyl- l -tryptophan, was able to restore the T-cell prolif-eration indicating an essential role of IDO inhibiting mecha-nism in IFN-γ- but not TNF-α-mediated suppression [ 35 ].

Under normal culture conditions, our blood-derived MSC line secreted cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ), expressed adhesion molecules on their surface (H-CAM, V-CAM-1, β1-integrin, and α4-integrin) as well as a wide range of chemokine receptors. Most of the expressed chemokine receptors belong to the group of ho-meostatic chemokine receptors [ 36 , 37 ], which are known to be involved in homeostatic leukocyte traffi cking. The con-cept of using MSCs as delivery vehicles in cellular therapy is based on the fact that tumors and other damaged sites send out chemoattractant signals in order to recruit MSCs to form the supporting stroma of those target tissues. MSCs delivery seems to be both safe and feasible and may be a promising approach for the delivery of therapeutic agents in the fu-ture. However, the recruitment of MSCs depends on signals from the target tissue microenvironment, and it is tempt-ing to speculate that the cell biological functions of MSCs will similarly be modulated by tissue-derived mediators. Understanding the factors and mechanisms regulating their homing are crucial, and could allow us to manipulate MSCs for therapeutic use. Therefore, the functionality of blood-derived MSC line surface-expressed chemokine receptors was demonstrated by migration assays. We demonstrated a migratory effect of the chemokine receptor ligands CCL5/RANTES (for CCR1, CCR3, and CCR5), CCL17/TARC (for CCR4 and CCR7), CCL21/6Ckine (for CCR7), CCL28/MEC (for CCR3 and CCR10), CXCL12/SDF-1α (for CXCR4), and CXCL13/BCA-1 (for CXCR5). These fi ndings support the notion that several CXC and CC chemokines play a key role in MSCs homing, localization, and recruitment to sites of tissue damage, infl ammation, and tumor as described

HEMEDA ET AL. 704

Author Disclosure Statement

No competing fi nancial interests exist.

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Acknowledgments

We would like to acknowledge Dr. Ralf Huss and Dr. Peter Nelson for kindly providing the V54/2 cell line and Kirsten Bruderek and Petra Altenhoff for their excellent technical assistance. This project was supported in part by the Else-Kröner Fresenius Stiftung and the German Israeli Foundation.

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HEMEDA ET AL. 706

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Address correspondence to: Prof. Sven Brandau

Department of Otorhinolaryngology University Hospital Essen

Hufelandstr. 55 Essen 45122

Germany

E-mail : sven.brandau@uk-essen.de

Received for publication September 17, 2009Accepted after revisions January 12, 2010

Prepublished on Liebert Instant Online January 12, 2010

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