AbstractMultipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14−CD1a+) and resulted in the generation of CD14+CD1a− cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an anti-inflammatory interleukin-10-producing cell type.
Multipotent stromal cells (MSC) are non-hematopoietic progenitor cells that have been identified in different tissues and are capable of differentiation towards adipogenic, osteogenic and chondrogenic lineages. Importantly, MSC have immunoregulatory properties. These properties have been the subject of many studies over the years and make MSC interesting candidates for further clinical application.
In several experimental disease models, MSC have been shown to modulate immune responses in vivo. Administration of MSC has been shown to prevent graft-versus-host disease1 and the development of experimental autoimmune encephalomyelitis in mice2 and results in prolonged skin graft survival in baboons.3 The use of MSC as a cellular therapy is currently being explored in clinical trials, including treatment of graft-versus-host disease,4 promotion of engraftment following hematopoietic stem cell transplantation5 and treatment of Crohn’s disease.6
The mechanisms underlying the immune suppression by MSC are still unclear. It has been widely shown that MSC suppress the proliferation of T cells upon allogeneic or mitogenic stimulation.7,8 Soluble factors proposed to be involved in this effect include indoleamine 2,3-dioxygenase, prostaglandin E2, transforming growth factor-β1, interleukin (IL)-6 and heme oxygenase 1.9–13 MSC also inhibit the proliferation of B cells and IL-2-stimulated natural killer cells14,15 and have been described to promote the formation of regulatory T cells (Treg).16–18
MSC can also modulate immune responses through inhibition of monocyte-derived immature dendritic cell (iDC) differentiation.19,20 Monocyte differentiation towards iDC can be inhibited by IL-6 and IL-1021,22 and several studies have suggested that IL-6 may play a role in the inhibitory effect of MSC on the differentiation of monocytes to dendritic cells.19,20 These data are, however, derived from in vitro co-culture experiments of MSC and monocytes in which the cellular source of cytokine production was not identified. The specific role of these cytokines in inhibiting MSC functionality is, therefore, still unclear.
In this study, we investigated the mechanism of inhibition by MSC during the differentiation of monocytes towards iDC and focused on the exact source of the cytokines involved. To this end we performed co-culture, transwell and supernatant-supplemented experiments of monocytes and MSC and applied neutralization and depletion strategies to reveal the cellular interactions that are crucial for the immunomodulating effect of MSC on monocyte differentiation.
Design and Methods
Generation of human multipotent stromal cells
Adult bone marrow was harvested from healthy donors or from orthopedic patients after obtaining their written informed consent, according to procedures that were approved by the medical ethical committee. Mononuclear cells were isolated and plated at a density of 1.3×10/cm in DMEM-low glucose (Invitrogen Corp., Paisley, UK) supplemented with 10% fetal calf serum (Greiner Bio-one) and penicillin/streptomycin (Invitrogen Corp.). When MSC reached confluency the cells were detached, characterized by FACS analysis and used in the co-culture experiments at passages 2–5. Further details are given in the Online Design and Methods.
Isolation and differentiation of monocytes
Human peripheral blood mononuclear cells from healthy donors were isolated from buffy coats obtained from Sanquin Blood Supply. CD14 monocytes were purified with a MACS LS column using CD14 microbeads according to the manufacturer’s recommendations (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).
Freshly isolated monocytes (CD14) were cultured at a concentration of 1.0×10 cells/well in 6-well plates in RPMI containing penicillin/streptomycin, L-glutamine (Invitrogen Corp.) and 10% fetal calf serum supplemented with the growth factors IL-4 (10 ng/mL) and granulocyte-monocyte colony-stimulating factor (GM-CSF) (5 ng/mL) (both from Invitrogen Corp.) for 6 days, resulting in the generation of iDC (CD14/CD1a). To examine the effect of MSC on monocyte differentiation, irradiated MSC (60 Gy) were added enabling direct cell-cell contact or in a transwell system (pore size 0.4 μM; Corning Inc., Lowell, MA, USA) at a MSC:monocyte ratio of 1:10, as described previously.23,24 In some experiments IL-10 (20 ng/mL), IL-6 (100 ng/mL), anti-IL-10 (2–20 μg/mL) or anti-IL-6 (2.5 μg/mL) (all from R&D systems Europe Ltd., Abingdon, UK) was added. The involvement of secreted factors was further assessed by addition of conditioned medium from MSC alone (MSC-CM), MSC cultured with growth factors (MSC+GF CM) and from the monocyte-MSC co-cultures (MSC+mono CM) to the monocyte differentiation culture. In some experiments, IL-6 was depleted from the MSC-CM using the μMACS Streptavidin kit (Miltenyi Biotec) according to the manufacturer’s recommendations. MSC-CM was used in the monocyte differentiation in a 2:1 ratio with fresh medium. Further details are given in the Online Supplementary Design and Methods.
The flow cytometry analysis is described in the Online Supplementary Design and Methods.
Analysis of cytokines
The concentrations of IL-6 and IL-10 were measured in cell-free supernatants collected on day 6 from cultures of monocytes with or without MSC. To distinguish between cytokines produced by MSC or monocytes, the cells from the transwell co-cultures were separated on day 6 and cultured further in fresh medium for an additional 2 days and IL-6 protein concentrations were determined. Further details are given in the Online Supplementary Design and Methods.
The mRNA expression of IL-6, IL-10 and β-actin was measured in MSC and monocyte populations on day 6 of the co-culture. All data were normalized using β-actin as a reference gene. Further details are given in the Online Supplementary Design and Methods.
Allogeneic mixed lymphocyte reaction assay
Monocytes cultured for 6 days in the presence or absence of MSC in a transwell co-culture were tested for their ability to stimulate the proliferation of allogeneic CD4CD25 T cells that were isolated from peripheral blood mononuclear cells by MACS using the CD4CD25 regulatory T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The CD4CD25 T cells were stimulated with iDC or the monocyte-derived cells from the monocyte-MSC co-culture at various ratios. After 4 days of incubation, cells were pulsed overnight with 0.5 μCi of [H]-thymidine to determine T-cell proliferation. Further details are given in the Online Supplementary Design and Methods.
All data represent the average and standard deviation from multiple MSC donors. Unless otherwise specified, statistical analyses were conducted using the Student’s t test for two groups and by a two-way ANOVA with a Bonferroni post-test for comparison of three groups. Prism5 software (GraphPad Software, Inc., CA, USA) was used.
Multipotent stromal cells skew the differentiation of monocytes to the formation of CD14+ cells with low allostimulatory capacity
Monocytes (purity after MACS isolation was more than 90% CD14 cells) cultured in the presence of IL-4 and GM-CSF differentiated into CD1aCD14 iDC. Following addition of MSC to these cultures, monocyte differentiation towards iDC was inhibited (Figure 1A,B). In the presence of MSC, a significantly lower fraction of monocytes acquired CD1a (15.1±7.4% versus 70.6±8.8%, P=0.002, n=7) and a significantly lower fraction lost CD14 expression (52.2±8.1% versus 99.5±0.1%, P=0.001, n=7) (Figure 1B).
The MSC were analyzed for expression of surface markers before and after co-culture with monocytes. MSC expressed HLA-class I, CD73, CD105 and CD90 and did not show expression of HLA-class II, CD31, CD45, CD80 or CD34. No changes were observed after co-culture with monocytes.
The monocyte-derived populations, formed in the absence or presence of MSC in transwell co-cultures, were functionally tested for their ability to induce proliferation of allogeneic T cells (Figure 1C). iDC effectively induced proliferation of allogeneic CD4CD25 T cells. The monocyte-derived population that was generated in the presence of MSC showed a significantly reduced allostimulatory capacity compared to the iDC.
The concentrations of both interleukin-6 and interleukin-10 are increased in co-cultures of monocytes and multipotent stromal cells
Monocytes were cultured in the presence of IL-4 and GM-CSF and in the presence or absence of MSC and cytokine concentrations were measured in cell-free culture supernatants of the direct co-cultures. Supernatants from monocytes that were cultured in the presence of MSC contained significantly higher concentrations of IL-6 and IL-10 than control supernatants derived from cultures without MSC (IL-6: 984.6±122.6 pg/mL versus 333.4±15.1 pg/mL, n=3, P<0.0001; IL-10: 221.6±30.0 pg/mL versus 11.9±4.4 pg/mL, n=3, P<0.05; Figure 2A,B).
Cytokine expression by multipotent stromal cells and monocytes
The MSC-induced changes in surface marker phenotype and cytokine concentrations in the monocyte cultures were also observed when the experiments were performed in a transwell system (data not shown). To assess which population was responsible for the observed increase in cytokine concentrations, the MSC and the monocyte-derived cell populations from such transwell cultures were assessed at day 6 for expression of IL-6 and IL-10 mRNA. Neither unstimulated MSC nor MSC cultured in the presence of IL-4 and GM-CSF and in the presence or absence of monocytes, were found to express IL-10 mRNA (Figure 2C). In agreement with this observation, IL-10 protein was not detected in culture supernatants from unstimulated MSC or from MSC cultured in the presence of IL-4 and GM-CSF (data not shown). Monocyte-derived cells, however, expressed detectable levels of IL-10 mRNA (fold change of 3.47×10±8.5×10 relative to β-actin) and these expression levels were considerably increased in the presence of MSC (P<0.05; Figure 2D). These data indicate that the IL-10 protein detected in the supernatants of monocyte-MSC co-cultures was produced by the monocyte-derived cell population.
Next, we analyzed expression levels of IL-6. Figure 2C shows that unstimulated MSC express IL-6 mRNA. A 5-fold increase in the average level of IL-6 mRNA expression was observed in MSC from cultures containing IL-4 and GM-CSF, but this increase did not reach statistical significance (P=0.06), which was probably due to the high variation that was observed between the different MSC samples. No further increase in IL-6 mRNA expression was observed in MSC that were co-cultured with monocytes. After the 6 days of co-culture, MSC and monocytes were separated and cultured for another 2 days to measure the cytokine concentrations secreted by both populations after co-culture. As for the IL-6 mRNA expression in MSC, the concentrations of IL-6 protein in culture supernatants of MSC cultured in the presence of IL-4 and GM-CSF and in the presence or absence of monocytes were not significantly increased compared to those in the culture supernatants of unstimulated MSC (Figure 2E).
IL-6 mRNA was also expressed in the monocyte-derived iDC. Significantly increased expression levels (P<0.001) were found in monocyte-derived populations that were generated in the presence of MSC (Figure 2D). Concordantly, the concentrations of IL-6 in the supernatant of monocytes cultured for an additional 2 days after the 6 days of MSC-monocyte co-culture were significantly increased compared to those of the control culture of monocytes with IL-4 and GM-CSF (P<0.05; Figure 2F).
Secreted factors from unstimulated multipotent stromal cells are responsible for the inhibition of monocyte differentiation
The observation that MSC also displayed their inhibitory effect on the differentiation of monocytes when the co-cultures were performed in a transwell system showed that cell-cell contact between monocytes and MSC was not required and that soluble factors were responsible for this effect. We investigated whether these soluble factors were secreted by culture-expanded MSC or only by MSC stimulated in the co-cultures. To do this, three types of conditioned medium were collected and tested for their ability to inhibit monocyte differentiation: (i) conditioned medium from unstimulated MSC (MSC-CM), (ii) conditioned medium from MSC cultured with the growth factors IL-4 and GM-CSF (MSC+GF CM) and (iii) conditioned medium from the monocyte-MSC direct co-cultures (MSC+mono CM). Figure 3 shows that the differentiation of CD1aCD14 monocytes into CD1aCD14 iDC was completely inhibited in the presence of CM derived from stimulated, but also from unstimulated MSC. This shows that MSC constitutively produce and secrete the essential factor(s) that cause their inhibitory effect on monocyte differentiation to iDC.
Role of interleukin-6 and interleukin-10 in the multipotent stromal cell-induced inhibition of monocyte differentiation
To study the exact role of IL-6 and IL-10 in the inhibition of monocyte differentiation by MSC, neutralizing antibodies against IL-6 and IL-10 were added to the transwell co-cultures. Addition of neutralizing antibodies against IL-10 did not result in restored iDC formation (Figure 4A). Addition of neutralizing antibodies against IL-6, however, caused a partial, but significant reduction of the inhibitory effect of MSC and resulted in increased CD1a expression and a decrease in the expression of CD14 on monocytes (Figure 4B,C). This neutralization of IL-6 substantially reduced the upregulation of IL-6 mRNA and IL-10 mRNA expression in the monocyte-derived population (Figure 4D) and reduced IL-10 protein concentrations (Figure 4E) in the monocyte cultures with MSC or MSC-CM.
We confirmed the observation that addition of either IL-6 or IL-10 resulted in inhibition of monocyte differentiation (Figure 5A-B). However, since MSC do not produce IL-10 (Figure 2), only IL-6 can be an MSC-derived factor that is relevant for the inhibitory effect of MSC. Indeed, we showed that addition of only IL-6 to the monocyte cultures induced IL-10 expression in monocytes, similar to the effect of MSC or MSC-CM (Figure 5A). To confirm that IL-6 is indeed the key factor produced by MSC, we depleted IL-6 from MSC-CM and studied the effect of this IL-6 depletion on monocyte differentiation. After depletion of IL-6, 84–96% of the IL-6 was depleted from the MSC-CM. Figure 5C shows that the inhibition of monocyte differentiation by MSC-CM was reversed by depletion of IL-6 from the CM. IL-6 depletion completely reversed the formation of a CD14 monocyte-derived population, but did not reverse the MSC-CM-mediated inhibition of CD1a upregulation. Functional testing revealed that the MSC-CM-induced IL-10 secretion in the monocyte cultures was completely abolished following IL-6 depletion (Figure 5D). The allostimulatory capacity, however, was not restored by depleting IL-6 from the MSC-CM (Figure 5E). Overall, our results show that MSC-derived IL-6 is an essential factor for the inhibition of monocyte differentiation by MSC and for inducing an IL-10-producing, monocyte-derived CD14CD1a cell population. The neutralizing and depletion experiments show that IL-6 is an important factor, but not the only one, which is produced by MSC to exert an inhibitory effect on monocyte differentiation.
It has been acknowledged that MSC inhibit the differentiation of monocytes towards iDC. The cytokines IL-6 and IL-10 have been described to play a role in the differentiation of monocytes towards iDC.21,22 However, the role of these cytokines in the MSC-induced inhibition of this process is still unclear. In this study, we used an MSC-monocyte co-culture system to identify the cytokines that are crucial in this process and reveal their exact cellular source of production. The addition of MSC to monocyte cultures containing IL-4 and GM-CSF inhibited the monocyte-derived iDC differentiation and significantly increased the concentrations of the cytokines IL-6 and IL-10. Transwell experiments showed that cell-cell contact was not required for the inhibitory effect of MSC on monocyte differentiation, indicating that soluble factors were responsible for this effect. Subsequent experiments with MSC-CM showed that unstimulated MSC produced the soluble factors responsible for inhibition of monocyte differentiation. Previous studies suggested that IL-6 plays a role in the inhibitory effect of MSC on the differentiation of monocytes to dendritic cells.19,20 Whether this is a direct or indirect effect remained unclear since both monocytes and MSC are capable of IL-6 production and secretion. We show that IL-6 production by MSC is constitutive and not significantly enhanced upon contact with monocytes. Since the effect of MSC could be replaced by MSC-CM, we considered MSC-derived IL-6 to be responsible for the MSC-induced inhibition of monocyte differentiation. Indeed, addition of neutralizing antibodies to IL-6 significantly reversed the inhibitory effect of MSC and depletion of IL-6 reversed the inhibitory effect of MSC-CM. These data clearly indicate that MSC-derived IL-6 regulates in vitro monocyte differentiation, thereby skewing monocytes towards an IL-10-producing cell type with reduced allostimulatory capacity. Additional factors might however play a role in the complete inhibition of monocyte differentiation towards iDC by MSC, since the allostimulatory capacity of the monocyte-derived cells was not restored by depleting IL-6 from the MSC-CM.
We found that, in spite of a number of reports making this claim,10,25–32 MSC do not produce IL-10, but that IL-10 is exclusively produced by monocytes in this system. Several reports claiming that the immunomodulatory properties of MSC are mediated by IL-10 have used antibodies to neutralize IL-10 in co-cultures of MSC and other cell types, including monocytes. These studies were not, therefore, designed to identify the cells that produce IL-10. Many reports indicate that MSC need to be activated in order to exert their immunomodulatory activities or to produce IL-10.26,33,34 Our data clearly show that activation of MSC is not required for the IL-10 induction in monocytes.
Activated monocytes have been shown to increase their IL-10 expression upon IL-10 exposure.35 Such increased IL-10 production could further enhance the immunomodulatory effect of MSC. Increased IL-10 secretion is a characteristic of type II activated macrophages, which have an immunomodulatory, anti-inflammatory function.36,37 Type-II-activated macrophages have also been shown to be able to induce regulatory T cells,38 another potential additional indirect immunomodulatory effect of MSC.
Taken together, we propose the hypothesis that MSC, by inducing IL-10 production in monocyte-derived cells, play a powerful regulatory role in multiple anti-inflammatory mechanisms (Figure 6), which could explain their clinical benefits in immunotherapy such as the treatment of graft-versus-host disease. Thereby MSC may act as potent modulators of the (innate) immune response inducing an anti-inflammatory environment.
- The online version of this article has a Supplementary Appendix.
- Funding This work received financial support from the Netherlands Organization for Scientific Research (NWO) ZonMW Translational Adult Stem Cell (TAS) program nr 11.600.1016.
- Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received September 19, 2012.
- Accepted January 11, 2013.
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