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Abstract

Cells derived from the amniotic membranes of human term placenta have drawn much interest for their characteristics of multipotency and low immunogenicity, supporting a variety of possible clinical applications in the field of cell transplantation and regenerative medicine. We have previously shown that cells derived from the mesenchymal region of human amnion (AMTC) can strongly inhibit T-lymphocyte proliferation. In this study, we demonstrate that AMTC can block differentiation and maturation of monocytes into dendritic cells (DC), preventing the expression of the DC marker CD1a and reducing the expression of HLA-DR, CD80, and CD83. The monocyte maturation block resulted in impaired allostimulatory ability of these cells on allogeneic T cells. In attempting to define the mechanisms responsible for these findings, we have observed that the presence of AMTC in differentiating DC cultures results in the arrest of the cells to the G₀ phase and abolishes the production of inflammatory cytokines such as TNF-α, CXCL10, CXCL9, and CCL5. Finally, we also demonstrate that the monocytic cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage. Taken together, our data suggest that the mechanisms by which AMTC exert immumodulatory effects do not only relate directly to T cells, but also include inhibition of the generation and maturation of antigen-presenting cells. In this context, AMTC represent a very attractive source of multipotent allogeneic cells that promise to be remarkably valuable for cell transplantation approaches, not only due to their low immunogenicity, but also because of the added potential of modulating immune responses, which could be fundamental both for controlling graft rejection after transplantation and also for controlling diseases characterized by inflammatory processes.

Keywords

Amnion Mesenchymal stromal cell Human placenta Cell transplantation Immunosuppression Dendritic cell Monocyte

Introduction

We and others have recently reported that cells derived from the amniotic and chorionic fetal membranes can engraft different tissues after xenogeneic (4) and allogeneic transplantation (34), which indicates the low immunogenicity of these cells. In addition, human amniotic cells are capable of modulating the immune response in vitro, and, in particular, are able to inhibit allogeneic lymphocyte proliferation, both when added in a contact (60) or a transwell setting (33,55). These results are in accordance with data reported for mesenchymal stromal cells (MSC) of other sources such as bone marrow (40), adipose tissue (35,44,60), and cord blood (14, 56). Based on their capacity to modulate immune responses, MSC have been proposed for clinical use in immune-mediated disorders (54) and encouraging results have been reported in clinical approach, including the treatment of graft-versus-host disease after allogeneic stem cell transplantation (29,49). Meanwhile, amniotic membrane has been clinically applied in wound healing (9,19), burn injuries (25), chronic leg ulcers (57,58), and in ophthalmology disorders (12,17) without acute rejection in the absence of immunosuppressive treatment. However the mechanisms involved in keeping the low immunogenicity of these cells and to control the inflammatory processes associated to some of these clinical conditions are still not completely revealed.

We have recently reported that amniotic mesenchymal tissue cells (AMTC) contain not only a stromal cell subpopulation (hAMSC) with typical MSC characteristics, such as positivity for CD105, CD73, and CD90 and absence of hematopoietic markers and of HLA-DR (42), but also a subpopulation with monocyte-macrophage phenotypic characteristics that are positive for HLA-DR, CD45, CD14, and CD11b. We have also demonstrated that both cell populations are of fetal origin (33). Even though the exact function of the latter of these two subpopulations is not yet clear, we have shown that these cells are capable of providing costimulatory signals to primed T cells (33), indicating that within the total amniotic mesenchymal population, which in toto exerts an antiproliferative effect, there exists a stimulatory cell subpopulation. These findings may help to reconcile controversial data that report both immunosuppressive and immunostimulatory effects of bone marrow-derived MSC (16,26,30,43). In particular, much scientific debate is centered on reports on the stimulatory effects of mesenchymal cells on T-cell proliferation and the capacity of these cells to immunomodulate allogeneic responses (47) both in in vitro and in vivo in the clinical setting of graft-versus-host disease suppression (49). Our finding of a stimulatory cell population in AMTC suggests that both inhibitory and stimulatory effects can be elicited by these cells, and possibly other MSC populations, based on the integration of the effects deriving from cells with opposing functional characteristics.

Although widely recognized, the mechanisms underlying the immunosuppressive effects of MSC are yet to be clarified. Recently, the effects of these cells on dendritic cell (DC) differentiation from peripheral blood monocytes (5,23,41,46,62), bone marrow (11,13), and CD34-positive cells (7,31,41,61) have been investigated. These studies have shown that MSC downregulate the expression of DC-related molecules such as CD1a, CD80, CD86, CD83, and HLA-DR during DC maturation. The mechanisms responsible for this inhibition remain to be elucidated, although several possible expla-nations have been proposed, including the involvement of soluble factors, such as IL-6 and M-CSF (7,8,11,23, 41), which are known to be produced by MSC and are important factors in the control of antigen-presenting cell development. Additionally, reduction of proinfiammatory cytokines such as interleukin-12, interferon-γ, and tumor necrosis factor-α (TNF-α), increased interleukin-10 and transforming growth factor-β (TGF-β) production (1,5,7,20,23,31,41,61,62), and induction of tolerogenic antigen-presenting cells have been described in cocultures of MSC with monocyte/DC/T cells. It has also been proposed that MSC can arrest monocytes in the G₀ phase of cell cycle through downregulation of cyclin D2 expression, thus inhibiting their differentiation into functional DC (46). Interestingly, several studies have shown that the inhibitory effects of MSC on DC differentiation are reversible after removal of MSC (23).

It is well known that DC are key regulators of immunity and tolerance (3), and that the stimulatory or tolerogenic function of DC depends on their maturation and activation state, in combination with the stimuli (cytokines) present in the microenviroment (32). Therefore, mechanisms that could control DC maturation and/or the cytokine microenviroment could also affect the chances of graft rejection after transplantation.

In this study, we have characterized the effects of cells derived from the mesenchymal region of amnion on differentiation and function of DC. We find that amniotic cells cause a clear block in maturation of monocytes into DC both in terms of phenotype and ability to stimulate T-cell responses. Interestingly, this inhibition does not seem to be completely reversible. We also show production of Th2 cytokines in cultures of monocytes stimulated toward DC differentiation in the presence of AMTC, while a Th1-related profile was observed in cultures differentiated in the absence of amniotic cells. Importantly, we also demonstrate that the monocytic-macrophagic CD14-positive cells present in the amniotic mesenchymal region fail to differentiate toward the DC lineage.

Materials and Methods

Isolation of Amniotic Mesenchymal Tissue Cells

Placentas were obtained from healthy women after vaginal delivery or caesarean section with informed consent according to the policy approved by the local Ethical Committee. Amniotic mesenchymal tissue cells (AMTC) were isolated from human term placenta as previously described (33). Briefly, the amnion was manually separated from the chorion, washed extensively in phosphate-buffered saline (PBS) (Sigma, St. Louis, MO, USA) containing 100 U/ml penicillin and 100 μg/ml streptomycin (both from Euroclone, Whetherby, UK), and cut into small pieces. Amnion fragments were incubated at 37°C for 8 min in PBS containing 2.4 U/ml dispase (BD Biosciences, San Jose, CA, USA), followed by a resting period of 10 min at room temperature in RPMI complete medium composed of RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine (Lonza). Afterwards, the fragments were digested with collagenase (0.75 mg/ml) (Roche, Mannheim, Germany) and DNAse (20 μg/ml) (Roche) for approximately 3 h at 37°C. Resulting cell suspensions were gently centrifuged (150 × g for 3 min) and the supernatant was filtered through a 100-μm cell strainer (BD Falcon, Bedford, MA, USA). Finally, AMTC were collected by centrifugation at 300 × g for 10 min.

The AMTC population contains two subpopulations of fetal origin, one of which is termed human amniotic mesenchymal stromal cells (hAMSC) (42), and the other of which contains cells positive for HLA-DR, CD45, CD14, and CD11b (33).

Isolation of CD14-Positive Cells From AMTC

Separation of CD14-positive cells from fresh AMTC was performed using the MACS system and direct labeling. Cells were first incubated with anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 20 min. After washing, enrichment of the CD14-positive subpopulation was performed by carrying out two subsequent column purifications using large cell columns (Miltenyi Biotec) according to the manufacturer's specifications. The percentage of CD14-positive cells was demonstrated by flow cytometry analysis to be greater than 90% of the total cells recovered.

Isolation of Monocytes and T Cells From Human Peripheral Blood Mononuclear Cells

Human blood samples were obtained from healthy donors with informed consent according to the policy approved by the local Ethical Committee. Human peripheral blood mononuclear cells (PBMC) were obtained through density gradient centrifugation (Lymphoprep, Axis Shield, Oslo, Norway) of heparinized whole blood samples.

Monocytes were obtained from PBMC through positive selection using anti-CD14-coated microbeads and Midi-Macs separation columns (Miltenyi Biotec) according to the manufacturer's instructions. The percentage of CD14-positive cells was demonstrated by flow cytometry analysis to be greater than 95% of the total cells recovered.

T lymphocytes were purified from PBMC through an indirect magnetic labeling system using the Pan T cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer's instructions. The percentage of CD3-positive cells was demonstrated by flow cytometry to be more than 95% of the total cells recovered.

Generation and Reinduction of Monocyte-Derived Dendritic Cells

Immature dendritic cells (iDC) were generated according to previously described protocols (5,39) by culturing 1 × 10⁶ monocytes in 1 ml of RPMI complete medium containing 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; kindly provided by Dr. Schweighoffer, Novartis, Austria) and 100 ng/ml interleukin-4 (IL-4; kindly provided by Dr. Schweighoffer) in 24-well plates at 37°C for 3–5 days (28). Development of mature dendritic cells (mDC) was then induced by adding 100 ng/ml lipopolysaccharide (LPS, Sigma-Aldrich) followed by incubation for 2 more days (28).

For AMTC cocultures, 0.4 × 10⁶ AMTC were added to monocyte cultures, and differentiation toward iDC and mDC was then induced (with AMTC/monocyte ratio of 1:2.5). We termed these cocultures [AMTC-iDC*] and [AMTC-mDC*], respectively (Table 1). Transwell chambers (0.4 μm pore size membranes, Corning Inc., Corning, NY, USA) were used to separate the monocytes from the AMTC and allow easy recovery of DC without AMTC contamination. In other coculture experiments, AMTC were added to differentiated iDC and maturation was then induced by adding 100 ng/ml LPS (Sigma-Aldrich) for 2 or 5 days. We termed these cocultures “iDC+AMTC+LPS 2 days” and “iDC+AMTC+ LPS 5 days,” respectively (Table 1).

Table 1.

Nomenclature Adopted

Name	Description
Monocyte	Monocytes before culture.
iDC	Monocytes cultured with GM-CSF plus IL-4 for 3–5 days.
[AMTC-iDC*]	Monocytes cultured with GM-CSF plus IL-4 for 3–5 days in the presence of AMTC, added at the beginning of the culture.
mDC	iDC cultured with LPS for 2 days.
[AMTC-mDC*]	Monocytes cultured with GM-CSF plus IL-4 for 3–5 days, and then maturated with LPS for 2 days. Culture carried on in the presence of AMTC, added at the beginning of the culture.
[AMTC-iDC*]+LPS	After culture of [AMTC-iDC*], the AMTC were removed and treated monocytes were washed extensively and activated with LPS for 2 days, in the absence of AMTC.
iDC+LPS+AMTC 2 days	Monocytes differentiated toward iDC (in the absence of AMTC) and then maturated with LPS in the presence of AMTC for 2 days.
iDC+LPS+AMTC 5 days	Monocytes differentiated toward iDC (in the absence of AMTC) and then maturated with LPS in the presence of AMTC for 5 days.
Rested mDC	mDC harvested, washed extensively, and replated in control medium (RPMI complete medium) for a resting period of 2 days.
Rested [AMTC-mDC*]	[AMTC-mDC*] washed extensively and replated in control medium for a resting period of 2 days, in the absence of AMTC.
Reinduced mDC	mDC after resting period, induced again towards DC differentiation, culturing them with GM-CSF plus IL-4 for 3–5 days and then maturated with LPS for 2 days.
Reinduced [AMTC-mDC*]	[AMTC-mDC*] after resting period, induced again towards DC differentiation, culturing them with GM-CSF plus IL-4 for 3–5 days, and then maturated with LPS for 2 days.

AMTC, amniotic mesenchymal tissue cells; GM-CSF: granulocyte-macrophage colony-stimulating factor; iDC, immature dendritic cells; IL-4: interleukin-4; LPS, lipopolysaccharide; mDC, mature dendritic cells.

For reinduction experiments, mDC and [AMTC-mDC*] were harvested, washed extensively in PBS, and replated in control medium (RPMI complete medium) for a resting period of 2 days. We termed these cultures “rested mDC” and “rested [AMTC-mDC*]” (Table 1). After the resting period, cells were plated at concentrations ranging from 0.5–1 × 10⁶ cells/ml in RPMI complete medium containing GM-CSF (100 ng/ml) and IL-4 (100 ng/ml) in 24-well plates and incubated for 3–5 days. The cells were then activated with 100 ng/ml LPS (Sigma-Aldrich) for 2 more days.

T-Cell Proliferation Assays

Monocytes, monocyte-derived cells that had been differentiated in the presence or absence of AMTC, and CD14-positive-AMTC before and after differentiation, were collected after culture and washed twice in PBS (Sigma). Different concentrations of these cells (ranging from 0.5 × 10³ to 50 × 10³) were then plated in RPMI complete medium, irradiated (3000 cGy), and added to 1.5 × 10⁵ responder T cells. All cultures were carried out in triplicate in round-bottom 96-well tissue culture plates (Corning), in a final volume of 150 μl of RPMI complete medium. T-cell proliferation was assessed after 5 days by adding [³H]thymidine (1 μCi/well, Perkin Elmer, Life Sciences, Zaventem, Belgium) for 16–18 h. Cells were then harvested with a Filtermate Harvester (Perkin Elmer) and thymidine incorporation was measured using a microplate scintillation and luminescence counter (Top Count NXT, Perkin Elmer).

Flow Cytometry Analysis

For evaluation of cell phenotype, cell suspensions were incubated for 20 min at 4°C with fluorescein isothiocyanate (FITC) or phycoerythrin or allophycocyanin-conjugated antibodies specific for human CD1a (clone HI149), CD14 (clone MϕP9), CD80 (clone L307.4), CD83 (clone HB15e), CD86 (clone 2331), and HLA-DR (clone TÜ36), or isotype controls IgG1 (clone X40), IgG2a (clone X39), IgG2b (clone MG2b-57). All monoclonal antibodies were obtained from BD Biosciences (San Jose, CA, USA) except for isotype control IgG2b that was purchased from Biolegend (San Diego, CA, USA). After incubation with antibodies, cells were washed with PBS containing 0.1% sodium azide (Sigma) and 0.1% bovine serum albumin (BSA) (Promega Corporation, USA), and were then analyzed with a FACSCalibur and the CellQuest Software (BD Biosciences). Dead cells were gated out by iodide propidium (IP) staining.

For intracellular protein and DNA analysis, cells were suspended in 90% ethanol overnight to denature proteins. Cells were then washed with PBS, incubated in 100 ng/ml FITC (Invitrogen, CA, USA) with IP/RNase staining buffer (BD Biosciences) for 30 min at 4°C, and acquired on a FACSCalibur (BD Biosciences).

Cytokine Assays

Monocytes cultured toward iDC and mDC in the absence or presence of AMTC, and iDC exposed to LPS in the presence of AMTC for 2 days, were harvested and centrifuged. The supernatant was then collected and frozen at −80°C for use in cytokine assays. Supernatants from 0.4 × 10⁶ AMTC, which had been cultured in transwell setting for 5 days, were also harvested and frozen.

The levels of TNF-α, IL-6, CXCL8/IL-8, CXCL10/IP-10 (interferon-inducible protein 10), CXCL9/MIG (monokine induced by interferon-γ), CCL5/RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), and CCL2/MCP-1 (monocyte chemoat-tractant protein 1) in cell culture supernatants were determined using a multiple cytometric beads array system (CBA-Th1/Th2 Cytokine kit II, and CBA-Chemokine kit I, both from BD Biosciences), according to the manufacturer's instructions. Samples were acquired with a FACSAria and analyzed with FCAP Array software (BD Biosciences).

Statistical Analysis

Analysis of variance (ANOVA) was used to asses the influence of treatment with amniotic cells and cell concentration on the different outcomes (T-cell proliferation, cytokine production) via linear regression model followed by Holm-Bonferroni adjustment method to correct for multiple comparison. ANOVA was performed after proper transformation of data by square root in order to stabilize variance. A treatment group by concentration interaction term was tested to investigate if treatment effect would change across different cell concentrations as well as a treatment group by cell type interaction. Values of p < 0.05 were considered statistically significant. Statistical analyses were performed using R software (version 2.7.0).

Results

Effect of AMTC on Differentiation of Monocyte-Derived Dendritic Cells

Isolated CD14+ monocytes were induced to differentiate into iDC through culture with GM-CSF and IL-4. Monocytes gave rise to iDC that expressed the typical dendritic cell marker CD1a in the absence of the monocytic marker CD14. Low expression of CD83, as well as expression of the costimulatory molecules CD80 and CD86, and HLA-DR were also observed (Fig. 1A, B). To study the effect of AMTC on DC differentiation, monocytes were differentiated with GM-CSF and IL-4 in the presence of AMTC (AMTC-iDC*) with AMTC/monocyte ratio of 1:2.5, which were kept physically separated using transwell membranes. This allowed study of the effects of AMTC in the absence of intercellular contact and easy recovery of monocyte-derived cells without AMTC contamination. Compared to cells stimulated in the absence of AMTC, the presence of AMTC strongly inhibited differentiation of monocytes into iDC. Indeed, cells stimulated under these conditions continued to express CD14 in the absence of CD1a (Fig. 1C), while CD80 and CD83 were absent or expressed at very low levels. CD86 and HLA-DR showed expression patterns similar to those observed for monocytes before induction of differentiation (Fig. 1C). In addition to these phenotypical modifications, monocytes differentiated in the presence of AMTC retained a more monocytic-macrophage morphology, showing a more rounded shape that lacks a veiled appearance (data not shown).

Figure 1.

Effect of AMTC on differentiation of monocytes to DC. Phenotypic analysis of isolated CD14⁺ monocytes (A) that were induced to differentiate into immature dendritic cells (iDC) with GM-CSF and IL-4 for 3–5 days in the absence (B) or presence (C) of AMTC from the beginning of the culture period. To induce differentiation toward fully mature DC (mDC), iDC and [AMTC-iDC*] were further cultured with LPS for 2 days (D, E). In some samples, AMTC were removed from the [AMTC-iDC*] cocultures before the stimulation with LPS (F). AMTC were also added for either 2 or 5 days during the LPS-induced maturation from iDC to mDC (G, H). After culture, expression of CD1a, CD14, HLA-DR, CD80, CD86, and CD83 was evaluated using flow cytometry by incubation with corresponding mAbs (open histograms) or isotype-matched antibodies (control, filled histograms). The results are representative of at least five individual experiments.

After exposure to LPS, iDC further differentiated into mDC, which was characterized by increased surface expression of CD83, strong positivity for the costimulatory molecules CD80 and CD86, and upregulation of HLA-DR (Fig. 1D). Exposure to LPS of monocytes cultured with GM-CSF and IL-4 in the presence of AMTC (AMTC-mDC*), resulted in lack of CD83 and CD80 upregulation, reduced CD86 and HLA-DR expression, and a final cell phenotype very similar to the original monocytes (Fig. 1E). We performed a titration of AMTC/monocytes (1:1, 1:2.5, 1:10), and observed that even at a 1:10 ratio a block of CD1a expression was maintained, while the expression of the costimulatory molecules CD80 and CD83 was drastically reduced but not completely abolished (data not shown).

To establish whether the observed block in maturation was due to the continuous presence of AMTC or to a particular state induced in monocytes by AMTC, we cultured monocytes with GM-CSF and IL-4 in the presence of AMTC and then, before LPS exposure, AMTC were removed, AMTC-treated monocytes were washed in PBS, and then activated with LPS in the absence of AMTC. Again, we observed that the monocytes failed to downregulate CD14 and upregulate HLA-DR, CD80, CD86, and CD83 (Fig. 1F).

To address whether AMTC could also affect the progression of DC from an immature status to a mature status, iDC were exposed to LPS in the presence of AMTC for 2 (Fig. 1G) or 5 days (Fig. 1H). Under these conditions, cells showed phenotypic characteristics very similar to mDC, with only a detectable decrease of CD83 expression that was mostly noticeable after 5 days of coculture (Fig. 1G, H). These findings indicate that AMTC have little effect on maturation of DC after these cells have become committed to immature DC.

Effects of AMTC on Protein Synthesis of Differentiating Monocytes

Recent data suggest that human MSC inhibit DC differentiation by preventing cell cycle entry and inhibiting protein synthesis in monocytes (46). To determine whether a similar mechanism may be induced by AMTC, we investigated the intracellular protein content of monocytes during differentiation to DC, both in the absence or presence of AMTC. The majority of monocyte-derived DC without AMTC had progressed to the G₁ phase, whereas monocytes induced in the presence of AMTC showed a almost complete arrest in the G₀ phase (Fig. 2). In contrast, addition of AMTC to iDC for 2 days during LPS exposure (iDC+LPS+AMTC 2 days) did not significantly affect the ability of these cells to progress to the G₁ phase (Fig. 2). However, consistent with the effects noted on the mDC phenotype (Fig. 1H), coculture of iDC and AMTC for 5 days during LPS induction (iDC+LPS+AMTC 5 days) resulted in a detectable reduction in the G₀-G₁ transition (Fig. 2).

Figure 2.

Effect of AMTC on protein synthesis during differentiation of monocytes to DC. Protein content (y-axis) and DNA analysis (x-axis) were analyzed by fluorescein isothiocyanate and propidium iodide staining, respectively, during monocyte differentiation into iDC and mDC in the absence or presence of AMTC. Representative FACS plots of at least three individual experiments are shown.

AMTC Affect the Ability of DC to Induce T-Cell Proliferation

Because AMTC were shown to inhibit monocyte-derived DC differentiation, we also wanted to determine whether the altered immunophenotype of the monocytes cultured toward iDC and mDC differentiation in the presence of AMTC corresponded to an alteration in the ability of these cells to stimulate T-cell proliferation. To this end, the different cell populations, namely iDC, [AMTC-iDC*], mDC and [AMTC-mDC*], were harvested, washed intensely, and used as stimulators in T-cell proliferation assays. Undifferentiated monocytes were able to induce T-cell proliferation in a dose-dependent manner (Fig. 3). Across all cell ratios that were tested, iDC and monocytes cultured toward iDC differentiation in the presence of AMTC showed a similar capacity to induce T-cell proliferation. In contrast, monocytes cultured toward mDC differentiation in the presence of AMTC induced a significantly lower level of T-cell proliferation, which was similar to that induced by monocytes cultured toward iDC in the absence or presence of AMTC (Fig. 3).

Figure 3.

AMTC affect the ability of DC to induce T-cell proliferation. T-cell proliferation induced by irradiated monocytes and monocyte-derived DC differentiated and matured in the absence or presence of AMTC. Different concentrations of irradiated stimulator cells were cocultured with 1.5 × 10⁵ allogeneic responder T cells. Lymphocyte proliferation was assessed by [³H]thymidine incorporation after 5 days of culture and expressed in cpm. The results shown are the average and SD of five individual experiments. There is no significant difference between the proliferation induced by iDC versus [AMTC-iDC*] and [AMTC-iDC*] versus [AMTC-mDC*], while proliferation induced by mDC is significantly higher than that caused by all the other stimulators at all concentrations tested. **p < 0.01; ***p < 0.001 mDC versus [AMTC-mDC*].

The Inhibitory Effect of AMTC on Monocyte Differentiation Is Not Completely Reversible

In order to understand whether the effect of AMTC on differentiation of monocytes toward DC was reversible, monocytes cultured toward mDC alone or in the presence of AMTC were harvested, washed, and replated in control complete medium in the absence of cytokines, LPS, and AMTC for a period of 2 days. After the resting period, the cells were reincubated with GM-CSF and IL-4 for 3–5 days, and then activated with LPS for a further 2 days. Both rested and reinduced cells were collected and analyzed by flow cytometry. Rested cells showed a similar profile to that observed before the resting period: mDC showed the typical profile characterized by CD1a expression and high levels of HLA-DR, CD80, CD86, and CD83 (Fig. 4A). On the contrary, AMTC-treated monocytes, after the resting period (rested [AMTC-mDC*]), expressed CD14 and not CD1a, as well as low levels of HLA-DR, CD80, CD86, and CD83 (Fig. 4A).

Figure 4A.

Incomplete reversibility of the effect of AMTC on DC differentiation. (A) Phenotypic analysis of monocytes induced to differentiate toward mDC in the absence or presence of AMTC after a resting period of 2 days in the absence of AMTC and after reinduction with GM-CSF, IL-4, and LPS. Expression of indicated markers was evaluated using flow cytometry by incubation with corresponding mAbs (open histograms) or isotype-matched antibodies (control, filled histograms). The results are representative of at least three individual experiments.

After GM-CSF and LPS exposure, reinduced mDC displayed the typical DC phenotype profile (Fig. 4A). In contrast, although the AMTC-treated monocytes after reinduction (reinduced [AMTC-mDC*]) showed down-regulation of CD14 expression and upregulation of CD80, these cells did not acquire the typical DC marker CD1a (Fig. 4A).

Next, we investigated the intracellular protein content of mDC and [AMTC-mDC*] after the resting period and reinduction. After the resting period, the cells showed a similar profile to that observed before resting: the majority of rested mDC were observed in the G₁ phase, whereas rested [AMTC-mDC*] were mostly retained in the G₀ phase (Fig. 4B). Instead, reinduced mDC and [AMTC-mDC*] showed a transition to the G₁ phase (Fig. 4B). Rested and reinduced cells were tested for their T-cell stimulatory properties. In all cases, cells that had been cultured in the presence of AMTC displayed reduced T-cell stimulatory capacities in comparison to controls (Fig. 4C).

Figures 4B and C.

(B) Protein content (y-axis) and DNA analysis (x-axis) were assessed by fluorescein isothiocyanate and propidium iodide staining, respectively, in monocytes induced to differentiate toward mDC in the absence or presence of AMTC after the resting period and reinduction. FACS plots representative of at least three individual experiments are shown. (C) T-cell proliferation was induced by coculturing 10 × 10³ of the irradiated indicated stimulator cells with 150 × 10³ allogeneic responder T cells. Lymphocyte proliferation was assessed by [³H]thymidine incorporation after 5 days of culture and expressed in cpm. The results are average and SD of five individual experiments. There is no significant difference between the proliferation induced by [AMTC-iDC*] versus [AMTC-mDC*], [AMTC-mDC*] versus rested [AMTC-mDC*], and rested [AMTC-mDC*] versus reinduced [AMTC-mDC*]. *p < 0.05; ***p < 0.001.

Monocytes Isolated From AMTC Fail to Differentiate to DC

Having shown that AMTC impair differentiation of monocytes into DC, we were interested in investigating whether the monocytic cells we recently identified within the AMTC (33) were also inhibited in their differentiation toward DC. As shown in Figure 5A, CD14-positive AMTC incubated with GM-CSF and IL-4 either alone or with LPS downregulate expression of CD14 but do not express the typical dendritic markers CD1a and CD83. They retain expression of the costimulatory molecules CD86 and HLA-DR, and do not acquire CD80 (Fig. 5A), indicating that these AMTC-derived CD14-positive monocyte/macrophage-like cells fail to differentiate toward the dendritic cell lineage. When tested for T-cell stimulatory potential, GM-CSF+IL-4+LPS-induced CD14-positive AMTC showed a significantly reduced ability to stimulate T cell proliferation compared to mDC (Fig. 5B).

Figure 5.

CD14-positive cells from AMTC are unable to differentiate into DC. (A) Phenotypic analysis of isolated CD14⁺ AMTC before and after culture with GM-CSF and IL-4 for 5 days (CD14⁺ AMTC+GM-CSF/IL-4) and LPS for 2 additional days (CD14⁺ AMTC+GM-CSF/IL-4+LPS). After culture, expression of indicated markers was evaluated using flow cytometry by incubation with corresponding mAbs (open histograms) or isotype-matched antibodies (control, filled histograms). The results are representative of at least four individual experiments. (B) T-cell proliferation induced by irradiated CD14-positive AMTC before and after culture with GM-CSF, IL-4, and LPS, irradiated monocytes, and monocyte-derived mature DC. Lymphocyte proliferation was assessed by [³H]thymidine incorporation after 5 days of culture and expressed in cpm. The results are average and SD of five individual experiments. There is no significant difference between the proliferation induced by monocytes versus CD14⁺ AMTC and CD14⁺ AMTC versus CD14⁺ AMTC+GM-CSF/IL-4+LPS, while proliferation induced by mDC is significantly higher than that caused by all the other stimulators at all concentrations tested. ***p < 0.001 CD14⁺ AMTC+GM-CSF/IL-4+LPS versus mDC.

Effect of AMTC on DC Cytokine Production

Because the cytokine microenviroment is critical for the recruitment, maturation, and function of DC, we next compared the levels of cytokines present in supernatants collected from cultures of AMTC as well as from iDC, mDC, monocytes cultured toward iDC or mDC differentiation in the presence of AMTC (AMTC-iDC* and AMTC-mDC*), and iDC matured with LPS in the presence of AMTC for 2 days (iDC+LPS+AMTC 2 days).

As shown in Figure 6, all cytokines tested were either not produced (TNF, IL-6, CXCL10/IP-10, CXCL9/MIG, CCL5/RANTES, CCL2/MCP-1), or were produced at very low levels (CXCL8/IL-8) by iDC, while an increase in the levels of all cytokines was observed after maturation of these cells to mDC.

Figure 6.

Effect of AMTC on DC cytokine production. The presence of TNF-α, CCL5/RANTES, CXCL10/IP-10, CXCL9/MIG, IL-6, CCL2/MCP-1, and CXCL8/IL-8 were quantified in supernatants from monocytes differentiated and matured toward DC in the absence or presence of AMTC, as well as from control AMTC culture supernatants. The results are average and SD of 10 individual supernatants for each cell population. *p < 0.05; ***p < 0.001.

AMTC did not produce TNF, CXCL10/IP-10, CXCL9/MIG, and CCL5/RANTES, while the presence of AMTC blocked the production of these cytokines by [AMTC-mDC*] cells. The presence of AMTC during the maturation phase resulted in a significant decrease in production of TNF and CXCL9, whereas the production of CXCL10/IP-10 and CCL5/RANTES was unaffected.

AMTC produce IL-6, and the presence of this chemokine was also observed in supernatants obtained from iDC or mDC, which had been differentiated in the presence of AMTC. Interestingly, although both AMTC and mDC produce high levels of IL-6, an additive effect in the levels of this chemokine was not observed when AMTC were added to iDC during LPS stimulation.

AMTC produced CXCL8/IL-8 at lower levels compared to those produced by mDC, and the addition of AMTC at the beginning of culture resulted in reduced levels of production of this cytokine by mDC. Meanwhile, the addition of AMTC during the maturation of iDC to mDC had no effect on the levels of CXCL8 observed.

AMTC also produced CCL2/MCP-1, at higher levels than those produced by mDC. We also detected significantly high level of this chemokine in all supernatant samples obtained in the presence of AMTC.

Discussion

In this study, we have shown for the first time that AMTC exert an inhibitory effect on monocyte differentiation into DC, and also cause a reduction in the ability of these cells to stimulate T-cell proliferation. Interestingly, removal of AMTC from monocyte cultures does not promptly reverse these inhibitory effects. We also demonstrated that the monocytic CD14-positive cell population that can be isolated from the amniotic mesenchymal region (33) fails to differentiate toward the DC lineage.

Several studies have recently shown that amniotic membrane-derived cells display immunomodulatory properties similar to those described for MSC derived from other sources, such as bone marrow, adipose tissue, and cord blood (42). Indeed, we and others have demonstrated that amniotic mesenchymal cells are able to modulate lymphocyte proliferation in mixed lymphocyte reactions, phytohaemagglutinin activation assays, and after activation via T-cell receptor cross-linking (33,60). Similar to other MSC, the mechanisms by which AMTC modulate immune responses are yet to be clarified, and the target cells onto which AMTC exert their immunomodulatory functions are largely unknown. Here, we have demonstrated that in addition to their known ability to act in vitro on T cells (33), AMTC also affect the generation of DC. DC are key regulators of immunity and tolerance, and their ability to initiate and regulate the response of T cells (i.e., cytotoxicity vs. tolerance) is closely related to the DC subtype and their maturation state (52). Importantly, our results demonstrate that monocytes that were exposed to DC differentiation and maturation conditions in the presence of AMTC showed impaired development, and failed to differentiate into CD1a⁺ DC, to upregulate the costimulatory molecules CD80, CD86, and CD83, and to increase surface expression of HLA-DR. Interestingly, addition of LPS to monocytes cultured toward iDC differentiation in the presence of AMTC did not result in differentiation, even after removal of AMTC, while addition of AMTC during the maturation step from iDC to mDC appeared to cause only downregulation of CD83 after 5 days of coculture, indicating that impairment of differentiation and maturation of monocyte-derived DC by AMTC occurs predominantly when the AMTC are added at the onset of the culture.

The mechanism that underlies inhibition of DC differentiation by AMTC involves arrest of the stimulated monocyte in the G₀ phase of the cell cycle. These data are consistent with a recent study by Ramasamy et al. (46) that focused on the effects of bone marrow MSC on cell cycle during monocyte differentiation and showed monocyte arrest in the G₀ phase. The AMTC-induced G₀ phase arrest appeared long-lasting and was persistent in monocytes cultured toward mDC differentiation in the presence of AMTC, and after the removal of AMTC and subsequent culture in fresh media for a period of 2 days, suggesting the hypothesis of a “blocking” condition; these cells were only observed to proceed to the G₁ phase after 5–7 days of culture under differentiation conditions in the absence of AMTC, although they did no acquire expression of CD1a.

Monocytes induced toward DC differentiation/maturation in the presence of AMTC showed a significant reduction in T-cell stimulatory capacity compared to mDC differentiated in the absence of AMTC. This reduced T-cell stimulatory capacity persisted even after removal of AMTC and complete reinduction of the cells toward dendritic cell differentiation, suggesting an irreversible functional effect of AMTC on differentiating monocytes. Previous studies on bone marrow MSC have reported that removal of MSC from MSC/DC cocultures is followed by a loss of CD14 positivity, but not by acquisition of CD1a expression, thus suggesting that the suppressive effect of MSC is not completely reversible (41). The question of reversibility of AMTC-induced inhibition is of critical importance for a complete understanding of the tolerogenic properties that AMTC may exert by interfering with the generation of antigen presenting DC, and this issue requires more extensive study.

Another open question regarding immunomodulatory properties of MSC concerns whether this process requires cell–cell contact. In our experiments using a transwell system, we observed that AMTC are able to inhibit monocyte differentiation in the absence of cell–cell contact, strongly suggesting the involvement of some soluble factor(s).

Several soluble mediators, such as TGF-β and hepatocyte growth factor (HGF) (10), prostaglandin E₂ (PGE₂) (1,53), indoleamine 2,3-dioxygenase (IDO) (27, 36,53), and, more recently, nitric oxide (NO) (48,51) have been reported to be involved in the inhibition of T-cell allogeneic response by bone marrow-MSC. In addition, IL-6 and M-CSF have also been reported to be produced by bone marrow MSC and may likely play a role in the modulation of DC differentiation (11,13,31,41).

Interestingly, we observed that AMTC produce high levels of the Th2-related cytokines CCL2, CXCL8, and IL-6, the last of which is known to inhibit the differentiation of CD34⁺ and monocytes into DC (8,38). In addition, we found that AMTC also block the production of the inflammatory cytokines TNF-α, CXCL10, CXCL9, and CCL5 in DC differentiation cultures, and the skewed Th2 cytokine secretion profile (with particularly high production of CCL2 and low production of TNF and CXCL9) was maintained in monocytes cultured toward mDC differentiation in the presence of AMTC after reinduction of differentiation (data not shown).

These results support the growing evidence indicating that one of the mechanisms responsible for the inhibitory properties of amniotic membrane-derived cells is the suppression of inflammatory processes, as already suggested previously from clinical experiences with burn injuries and wound healing (9,19,25), ophthalmology applications (12,17), chronic leg ulcers (57,58), and in animal models for spinal cord injury (50), drug-induced lung fibrosis (6), and ischemic stroke (24).

The fact that AMTC block production of CXCL10, a chemokine reported to promote and amplify allograft rejection (2,15,21), also reinforces the evidence that these cells may play an important role when cotransplanted for improvement of cell engraftment and for prevention of acute graft-versus-host disease. The induction of tolerance toward allogeneic grafts remains one of the major challenges in transplantation medicine, and amniotic membrane-derived cells may constitute promising candidates to be used to promote acceptance of transplanted organs due to their immunomodulatory and protective paracrine actions.

Finally, we demonstrate that the CD14-positive cells that we identified within the AMTC population, and that we recently reported to act as stimulators rather than inhibitors of T-cell proliferation (33), could not be induced to differentiate in vitro toward iDC or mDC. This block of DC differentiation resembles that induced by AMTC on monocyte cultures stimulated with DC-inducing cytokines. Due to the irreversible nature of the AMTC-induced inhibition that we observed in our experiments, it is tempting to speculate that these inhibited amniotic monocytes may contribute to fetal–maternal tolerance in the placental environment. Whether or not these inhibited monocytes are the same cells described as alternative activated macrophages, which have been described within the placental villi and which are thought to be involved in fetal tolerance (18), remains to be established.

Importantly, it is well accepted that the cytokine environment is crucial during pregnancy for the successful maintenance of fetal tolerance. In particular, it has been suggested that a Th2-like pattern of cytokine production (similar to that sustained by AMTC in culture) is necessary for a successful fetal outcome, while a Th1-like response is associated with an increased likelihood of fetal loss (22,37,45,59). If amniotic membrane-derived cells participate in the process of fetal–maternal tolerance is an unproven, but fascinating hypothesis.

In conclusion, our findings show that AMTC block the generation and maturation of antigen-presenting cells and abolish production of inflammatory cytokines, promoting a Th2-like cytokine profile. We speculate that their presence in the amniotic mesenchymal region is responsible for the inhibition differentiation of resident monocyte-like cells toward DC. Although our in vitro results may not be extrapolated to in vivo cell therapy applications, it is tempting to speculate that the effect of AMTC on T cells and DC could be exploited to obtain potent anti-inflammatory and immunosuppressive effects after transplantation. Previous findings and our current results indicate that amniotic-derived cells may constitute an important cell source for regenerative medicine and therapeutic approaches of immune-mediated disease (e.g., graft-versus-host disease, rheumatoid arthritis, Chron disease, multiple sclerosis) due to their engraftment, immunomodulatory properties, and protective paracrine effects. Further studies are warranted to verify and define the ultimate potential of these cells in vivo.

Footnotes

Acknowledgments

The authors thank the physicians and midwives of the Department of Obstetrics and Gynecology of Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy, and all of the mothers who donated placenta as well as all the volunteers who donated blood. The authors wish to thank Dr. Verzeletti, Dr. Alessi, and Dr. Ballini (Istituto Clinico St. Anna, Brescia, Italy) for the assistance with cell irradiation. The authors are indebted to Dr. Fabio Candotti for the support in data interpretation and critically reviewing the manuscript. The authors thank Dr. Marco Evangelista for help in editing the manuscript. This study was supported by grants from Fondazione Cariplo, Progetto Nobel.

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Amniotic Mesenchymal Tissue Cells Inhibit Dendritic Cell Differentiation of Peripheral Blood and Amnion Resident Monocytes

Abstract

Keywords

Introduction

Materials and Methods

Isolation of Amniotic Mesenchymal Tissue Cells

Isolation of CD14-Positive Cells From AMTC

Isolation of Monocytes and T Cells From Human Peripheral Blood Mononuclear Cells

Generation and Reinduction of Monocyte-Derived Dendritic Cells

T-Cell Proliferation Assays

Flow Cytometry Analysis

Cytokine Assays

Statistical Analysis

Results

Effect of AMTC on Differentiation of Monocyte-Derived Dendritic Cells

Effects of AMTC on Protein Synthesis of Differentiating Monocytes

AMTC Affect the Ability of DC to Induce T-Cell Proliferation

The Inhibitory Effect of AMTC on Monocyte Differentiation Is Not Completely Reversible

Monocytes Isolated From AMTC Fail to Differentiate to DC

Effect of AMTC on DC Cytokine Production

Discussion

Footnotes

Acknowledgments

References