MSC–DC interactions: MSC inhibit maturation and migration of BM-derived DC



Cytotherapy (2007) Vol. 9, No. 5, 451  458

MSC DC interactions: MSC inhibit maturation and migration of BM-derived DC Y-J Jung1, S-Y Ju2, E-S Yoo2, SJ Cho2, K-A Cho3, S-Y Woo3, J-Y Seoh3, J-W Park1, H-S Han4 and K-H Ryu2 1

Department of Microbiology, Gachon Medical School, Incheon, Korea, Departments of 2Pediatrics and 3Microbiology, Ewha Womans University College of Medicine, Seoul, Korea, and 4Department of Surgery, Seoul National University College of Medicine, Seoul, Korea

Background Mesenchymal stromal cells (MSC) comprise one of the BM stromal cells that are known to support hematopoiesis. It has also been suggested recently that MSC display immunosuppressive capacities through inhibiting the differentiation of monocyte-derived DC. DC travel to the lymph nodes (LN) to present Ag to T cells, and CCL21 is the chemokine that plays an important role in DC migration into the T-cell area of LN. We addressed the effect of MSC on this chemotactic activity of DC, one of the typical characteristics upon maturation. Methods BM cells were isolated and then cultured for generation of myeloid DC in the presence of GM-CSF and/or lipopolysaccharide with or without MSC. MSC were identified by flow cytometry of the immunologic markers and by performing colony-forming unit fibroblast assay. Migration of DC was observed with a newly developed time-lapse video microscopic technique.

Introduction BM is a complex tissue that contains hematopoietic stem cells in close contact with stromal cells, and this constitutes the BM microenvironment [1]. The BM stroma contains a small number of mesenchymal stromal cells (MSC) that have the capacity to form different mesenchymal cells, such as osteocytes, chondrocytes, adipocytes, tenocytes and skeletal myocytes, under appropriate conditions [2,3]. These properties of MSC have generated substantial interest for clinical application. Another great potential therapeutic use for MSC has arisen from the observation that they can exert an immunosuppressive effect. Studies conducted in human and animal models have demonstrated

Results MSC co-culture inhibited the initial differentiation of DC, as well as their maturation. The matured DC actively migrated directionally in response to CCL21, a powerful DC-attracting chemokine, whereas the MSC co-cultured DC did not. Discussion Collectively, the findings of these experiments raise the possibility that MSC suppress the migratory function of DC and so they may serve immunoregulatory activities through the modulation of the Agpresenting function of DC. Keywords chemotaxis, dendritic cells, maturation, mesenchymal stromal cells.

that MSC can inhibit T-cell proliferation in response to stimulation by allo-antigen and polyclonal mitogens [4  6]. Furthermore, intravenous administration of MSC has led to a modest, but significant, prolongation of skin graft survival in a pre-clinical baboon model [7]. The immunosuppressive effect of MSC has generally been ascribed to its inhibitory effect on T-cell proliferation [4]. However, the induction of immure responses is a complicated interaction between T cells and DC, and it has been shown recently that MSC have inhibitory effects on DC [8  10]. DC are the most potent APC and they are highly specialized in priming a T-cell-dependent immune response [9,11]. The hallmarks of DC are the ability to

Correspondence to: Kyung-Ha Ryu, Department of Pediatrics, College of Medicine, Ewha Woman’s University, Mok-dong 911-1, Yangchun Gu, Seoul, 158-710, Korea. E-mail: [email protected]. – 2007 ISCT

DOI: 10.1080/14653240701452057

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capture Ag, process and present antigenic peptides and migrate from the tissues they are distributed in to secondary lymphoid organs, where stimulation of naive T cells takes place [12,13]. The immature, Ag-processing DC are localized at diverse anatomical sites and they are incapable of effectively priming T cells [14  17]. Upon interaction with immune or inflammatory signals, DC rapidly acquire an activated phenotype and then enter into the circulatory system to home in on the T-cell areas of lymphoid organs. These mature DC have a very efficient T-cell-priming ability [13,18]. Thus the localization and trafficking of DC are important properties closely related with their maturation status. In the present study, we investigated the effect of MSC on the differentiation of mouse BM-derived DC with respect to their phenotype and chemotactic activities. This study has demonstrated that MSC remarkably reduce the chemotactic responsiveness and phenotypic characteristics of mature DC.

Methods Mice The animal care committee at Ewha Woman’s University College of Medicine (Seoul, Korea) approved all the procedures and protocols used in this study. Six to 8-week-old C57BL6 mice (Koatec, Pyeing Taek, Korea) were housed in the animal care facility, with food and water available ad libitum; they were exposed to a 12:12 h light  dark cycle in room air at room temperature.

incubation for 2  3 min at 378C with 0.25% trypsin solution (Invitrogen Life Technologies). Trypsin was neutralized by the addition of fresh complete medium. The resulting suspension was then expanded by plating it onto a new culture flask. Before experimental use, the MSC were tested for their ability to form colony-forming unit fibroblasts (CFU-F). CFU-F formation was visualized by Giemsa staining. The identity of the MSC was also confirmed by immunophenotypic criteria according to the following method of FACS analysis.

Differentiation of mouse myeloid DC The method for generating BM DC with GM-CSF was adapted from a previous publication [19]. BM cells were isolated as mentioned above and then plated in RPMI-1640 (Gibco BRL) supplemented with 10% FBS (Gibco BRL) at a concentration of 1 106/mL; 20 ng/mL rmGM-CSF (R&D, Minneapolis, NM, USA) were added to the cultured cells at days 0, 3, 6 and 8 to induce immature DC (imDC). Some of these cells were co-cultured with pre-established MSC during imDC generation period. For complete maturation, at day 10 the non-adherent cells were collected by gentle pipetting, centrifuged at 1200 r.p.m. for 5 min and resuspended with fresh media that contained 10 ng/mL rmGM-CSF and lipopolysaccharide (LPS; Sigma, St Louis, MP, USA) at 1 mg/mL (mDC generation). The cells were then cultured for 48 h. Some imDC were LPS stimulated in the presence of pre-established MSC.

FACS analysis Murine MSC isolation and culture expansion The mice were killed by cervical dislocation, and their femurs and tibiae removed and cleaned of all connective tissue. BM cells were collected by flushing the femurs and tibiae with RPMI-1640 media (Gibco BRL, Carlsbad, CA, USA) using a 25-gauge needle; the BM cells were filtered and washed by centrifugation at 1200 r.p.m. for 5 min. To initiate MSC culture, cells were plated on 25  75-cm2 flasks at concentrations of 5 106/mL/cm2 nucleated cells in a-modified Eagle’s medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen Life Technologies) and incubated at 378C in a 5% CO2 atmosphere. After 48 h, the non-adherent cells were removed by washing with 1 PBS and fresh medium then added. The medium was changed weekly. When the culture was near confluence, the monolayer cells were washed twice with 1PBS and then lifted by

Aliquots of cultured MSC and DC were stained using a panel of FITC- and PE-conjugated MAb for 30 min at 48C: for MSC, FITC anti-mouse CD106, FITC anti-mouse CD34, FITC anti-mouse CD31, PE anti-mouse CD73, PE  anti-mouse CD105 and PE  anti-mouse CD34; for DC, FITC anti-mouse CD11c, FITC anti-mouse CD40, FITC anti-mouse CD80 and PE  anti-mouse CD86. All the Ab were purchased from BD Pharmigen except for PE  anti-mouse CD105 (R&D). The cells were fixed with 1% paraformaldehyde solution and then analyzed with a FACS Calibur (BD Bioscience, San Jose, CA, USA).

mRNA detection by RT-PCR Total mRNA was extracted from DC. One microgram of mRNA was reverse-transcribed using a reverse transcription system (Promega, Madison, WI, USA) and the complementary DNA was amplified by using Taq DNA

MSC inhibit migration and maturation of DC

polymerase (Takara, Shiga, Japan) as follows: an initial denaturation step (at 948C for 3 min), 35 cycles of PCR (958C for 30 s, 508C for 30 s, 728C for 15 s) then a final step at 728C for 10 min, using the Gene Amp PCR system 9700 (Perkin Elmer, Norwalk, CT, USA). The following primers were used for amplification: mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5?-GTCTTCTCCACCATGGAGAAGGCT-3? and 5?-CATGCCAGTGAGCTTCCCGTTCA-3?; mouse CCR1, 5?-TCCTTCTCATCAGCAAGCTGTC-3? and 5?GAGGCAGCCCAGGTCCTTGAAG-3?; mouse CCR7, 5?-AAGAAACTGAGAAGCATGACG-3? and 5?-GAAACTGGAACACAACCACC-3?. The amplified DNA products were electrophoresed on 1.2% agarose gels and then stained with 0.5 mg/mL ethidium bromide.

Chemotaxis assay using time-lapse video microscopy The in vitro chemotactic activities of the DC were analyzed with a time-lapse video microscopic technique using special equipment (EZ-TAXIScanTM; Effector Cell Institute, Tokyo, Japan) [20]. The chemokine recombinant mouse CCL21 was purchased from R&D Systems. Six microchannels (6 mm depth) were created between a 10% FBS-coated cover slip and a silicon chip by assembling the holder set of the equipment according to the manufacturer’s instructions. Then 1 mL cell suspension that was

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resuspended at 1 106/mL in RPMI-1640 with 0.5% endotoxin-free BSA was injected into each well at one side of the microchannel, and 1 mL CCL21 (100 mg/mL) injected into the well at the other side of the channel. After the addition of CCL21 in the opposite compartment, the assay was performed for 120  180 min at 378C and the migratory behavior of the cells was recorded by a CCD camera every 30 s. After the assay, the digital images were converted into movies for analysis, and the number of the cells moving into the assay field was analyzed using the software (TAXIScan Analyzer, Effector Cell Institute) provided by the manufacturer.

Results Tissue culture planted BM cells differentiated into MSC MSC were isolated from BM, which contained heterogeneous populations of hematopoietic cells (Figure 1A), and then purified via adherence separation culturing. The MSC with short stick shapes adhered to the culture plates within 24 h (Figure 1B). At day 21, the spindle-shaped MSC proliferated into a layer (Figure 1C). After 14 days, the cells were quantified using a CFU-F assay (Figure 1D). Immunophenotypic analysis also showed that the cultured cells manifested the typical MSC surface phenotypes. At day 21, the surface phenotype was defined as being

Figure 1. Tissue culture-plated BM cells were differentiated into MSC. The newly plated BM cells (A) had a short stick shape after 24 h (B) and they proliferated into spindle-shaped adherent cells at day 21 (C). These cells also showed a capacity to form CFU-F at day 14, according to Giemsa staining (D). For the phenotypic markers, the MSC markers (CD73, CD105 and CD106) were increased, whereas the hematopoietic (CD34 and CD45) and endothelial cell markers (CD31) were not detectable at day 21 of the cultured cells (E).

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positive for CD73, CD105 and CD106 and negative for CD34, CD45 and CD31 (Figure 1E).

The addition of MSC suppressed the typical morphologic phenotypic characteristics of DC maturation induced by LPS Mouse BM cells differentiate into imDC in the presence of GM-CSF (20 ng/mL), with (MSC-imDC) or without MSC co-culture, according to a previous report [19]. After 10 days of imDC generation, LPS was added for another 48 h to stimulate the maturation of DC (mDC). Some of the imDC were LPS stimulated with MSC, and the MSC-imDC were LPS stimulated without MSC. After exposure to LPS, the relatively round-shaped imDC (Figure 2A) acquired the cell size and actively protruding cytoplasm that are typical morphologic characteristics for DC (Figure 2B). In parallel, the surface molecule phenotype of imDC (Figure 2E) showed the up-regulated expression of the co-stimulatory molecules CD86, CD80 and CD40 (Figure 2F). The addition of MSC during either the differentiation period or maturation period affected the morphologic and surface phenotypic characteristics of the DC. The mDC with the added MSC acquired the typical DC size; however, these cells were rather round and lacked the dendritic appearance (Figure 2C, D). In

these cells, the expression of CD86 was remarkably reduced compared with that of the mDC, and the CD80 and CD40 expressions were also slightly decreased (Figure 2G, H).

MSC co-culture induced CCR1 mRNA expression in the mDC Before activation, DC reside in peripheral tissues and the chemokine receptors expressed by DC include CCchemokine receptor (CCR)1, CCR2 and CCR5. These DC acquire a migratory capacity upon uptake of Ag. A key step in this process is the up-regulation of CCR7, which promotes DC migration into the T-cell areas of the draining lymph nodes [21]. Thus the migration of DC and the pattern of chemokine receptor expression depends on their maturation state. Because in the presence of MSC the maturation of imDC by LPS was observed to be inhibited, we examined whether MSC affect chemokine receptor expression on DC. LPS stimulation induced an increased expression of CCR7 in the DC even in MSC coculture (Figure 3). CCR1, a chemokine receptor involved in the peripheral tissue distribution of DC, was only observed in the mDC co-cultured with MSC (Figure 3). In our culture system, no CCR1 expression was detected in the DC with an immature status.

Figure 2. LPS stimulation induced large cytoplasmic dendrites (B, arrow head) from imDC having small protrusions (A). The development of dendrites from LPS-stimulated cells in the presence of MSC was not remarkable (C). This finding was similar to that of cells co-cultured with MSC first for 10 days even when LPS stimulation was done without MSC (D). The decreased expression of CD11c and co-stimulatory molecules in MSC-co-cultured mDC was compatible with morphologic observations. (E) imDC, (F) mDC, (G) mDC with MSC co-cultured during LPS stimulation; (H) mDC with MSC co-culture during imDC generation.

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for 10 days of the immature cell generation period (Figure 5D) than that of the MSC co-cultured for just 48 h of stimulation time (Figure 5C). The start of migration was faster (less than 10 min) for the LPS-stimulated DC than for the MSC-co-cultured DC groups (Figure 5B).

Discussion

Figure 3. Expression of chemokine receptor CCR1 on MSC cocultured DC. The RT-PCR products from the transcripts of CCR1, CCR7 and GAPDH were 383 bp, 383 bp and 420 bp, respectively. GAPDH RT-PCR was conducted for the loading control. Lane 1, fX174 HaeIII marker; lane 2, imDC; lane 3, mDC; lane 4, mDC with MSC co-culture during LPS stimulation; lane 5, mDC with MSC co-culture during imDC generation.

MSC prevented the directional migration of mDC in response to CCL21 on time-lapse microscopy As the chemokine receptor expression was altered by the addition of MSC during DC stimulation, we examined the responsiveness of DC to CCL21, a chemokine that acts on the migration of mature DC, by conducting a time-lapse video microscopic assay. The DC stimulated with LPS migrated directionally in response to CCL21 (Figure 4B and supplementary Figure 1B), whereas the unstimulated imDC did not (Figure 4A and supplementary Figure 1A). In the case of MSC co-culture, although the mDC migrated directionally to CCL21, the extent of migration was less than that of the mDC (Figure 4C, D and supplementary Figure 1C). Cellular migration in the KK microchamber was analyzed by depicting the kinetic change of the total cell counts (Figure 5). The decrease of CCL21 responsiveness was more prominent in the DC co-cultured with MSC

In the present study we have shown that MSC prevent the migration of mature DC and might modulate the expression of chemokine receptors. Several studies have indicated that MSC could exert an immunosuppressive effect in vitro . It has been demonstrated that MSC inhibit the proliferation of T cells stimulated by cognate Ag stimuli and non-specific mitogenic stimuli [4,6,22]. It has also been demonstrated recently that MSC inhibit the differentiation, maturation and activation of co-cultured DC [8,9]. Although precursor and immature DC are localized at diverse anatomical sites, upon exposure to inflammatory signals DC enter the circulatory system to home in on the T-cell areas of lymphoid organs, as well as undergoing functional maturation [12,23,24]. Thus trafficking of DC is a peculiar property of this cell group, in addition to their maturation-related morphologic and surface molecular changes. In this study, light microscopic examination as well as flow cytometry assay of the LPS-stimulated mDC without MSC co-culture showed the morphologic and phenotypic characteristics of the mature DC (Figure 2B, F). These cells displayed actively protruding dendritic cytoplasm and, in parallel, an increased expression of co-stimulatory molecules. However, the mDC co-cultured with MSC lacked these morphologic characteristics (Figure 2C, D) and the expression of surface markers of mDC (Figure 2G, H). As the DC generated in the presence of MSC have shown impaired responses to the signals that induce maturation [9,10], our observations correspond with the inhibitory effect of MSC on DC maturation. Naive T cells express CCR7 and the ligand for which CCL21 is expressed in lymphoid organs, therefore the circulation of naive T cells is directed into lymphoid tissues [25,26]. Upon maturation induced by danger signals, the expression of CCR7 is also up-regulated in DC [27,28], thus mature DC are promoted to migrate to the T-cell areas of the draining lymph node, where Ag presentation and subsequent T-cell activation take place. In our study, CCR7 mRNA was induced in all the LPSmatured DC, although the density of this expression was

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Figure 4. Time-lapse microscopic observation of the directional migration of imDC (A) and mDC (B  D) in a KK microchamber in response to CCL21. The mDC began to move vigorously toward the direction of the chemokine gradient, just like an amoeba crawls (B). On the other hand, the MSC-co-cultured mDC (C, D) showed only a slight migratory response compared with the response of mDC to CCL21. The CCL21 responsiveness of imDC (A) was unremarkable. (C) mDC with MSC co-cultured during LPS stimulation; D) mDC with MSC co-cultured during imDC generation.

slightly less in the MSC co-cultured group of DC than the LPS-only stimulated DC (Figure 3). In contrast, CCR1 was expressed in the MSC-co-cultured DC even with LPS stimulation (Figure 3). This observation is not in agreement with a previous study that showed down-regulation of CCR1 mRNA in DC after LPS activation [29]. As imDC express CCR1 and CCR5, which accounts for the chemotactic response to ligands of peripheral tissues [30], it is suggested that MSC hamper the migration of DC from peripheral tissues into draining lymph nodes even in the presence of immune or inflammatory signals. For the LPS stimulation-induced CCR7 expression in DC, regardless of MSC co-culture and CCR1 expression only on the MSC-co-cultured DC, we wonder whether

alterations of the chemokine receptor expression by MSC affect the chemotactic responsiveness of DC to CCL21, which is a ligand for CCR7. Very recently, a novel optical chemotactic assay has become available that provides microchannels in which stable concentration gradients of chemokines can be maintained [20]. In this assay, visual observation of the real cellular motions is very helpful for determining appropriately the chemotactic activities of the cells, including the migratory patterns and their extent, etc. The LPS-stimulated DC showed an early start of migration (Figure 5B) and demonstrated vigorous directional migratory activity to the CCL21 (Figure 4B and supplementary Figure 1B). The DC that were LPS stimulated in the presence of MSC also manifested

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Figure 5. Kinetic change of total cell counts in the assay field of the KK microchamber during chemotactic assay of imDC (A) and mDC (B  D) in response to CCL21. (C) mDC with MSC co-cultured during LPS stimulation; D) mDC with MSC co-cultured during imDC generation.

CCL21 responsiveness; however, the extent of their migration was reduced more than that of the fully matured DC (Figure 4C and 5C). For the DC with MSC co-culture for 10 days of the immature cell generation period, not only was the start of migration retarded (Figure 5D) but the number of migrating cells was remarkably decreased (Figure 4D and supplementary Figure 1C). It is well established that CCL21 has a central role in regulating the migration of tissue-resident DC to lymphoid organs. In addition, it has been suggested recently that a new and pivotal function of CCL21 is to drive the full maturation of inflammatory signal-licensed DC. Besides directing licensed DC to the draining lymphoid tissues, CCL21 provides further maturation signals that complete the DC maturation process and result in full T-cell activation [21]. Thus it can be speculated that MSC-induced hampering of DC migration acts synergistically on the maturation of DC together with the previously reported inhibitory effect of MSC on DC maturation [8 10], even under inflammatory conditions. In summary, our study suggests that MSC might promote immunoregulatory activities by not only acting directly on

the inherent maturation process of DC but also by inhibiting maturation-induced chemokine-receptor interactions.

Acknowledgements This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0520100) and Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R01-2006-000-10059-0).

Online supplementary material Supplementary Figure 1 shows the moving picture of migrating DCs in response to CCL21 on time-lapse microscopic system. It is available at http://www.informaworld. com/mcyt

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