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Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

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Related in: MedlinePlus

MSC-dependent reduction in CD25 expression by T-cells is mediated by MMP-2 and MMP-9. A: Enzymatic activities of MMP-2 and MMP-9 in medium conditioned by MSCs were analyzed by zymography. Conditioned medium was electrophoresed under nonreducing conditions and without heating through a 8% SDS-PAGE containing 0.1% gelatin. Following electrophoresis, the gels were incubated overnight in developing buffer containing SB-3CT to inhibit MMP activity. Gels were stained with Coomassie Blue, and densitometric quantification of MMP-2 and MMP-9 was performed using Scion software. Data were obtained from three individual experiments, and inhibition of MMP-2 and MMP-9 was determined by setting the mock treatment as 100%. □, control; ■, SB-3CT. B: To block activities of MMP-2 and MMP-9 in the MSC/T-cell coculture, SB-3CT was added at a concentration of 6 μmol/l. Stimulated T-cells alone (black curve) or cocultured with MSCs (dashed curve) or MSCs treated with SB-3CT (gray curve) were examined for surface expression of CD25. C: Stimulated T-cells were either cultured alone (black curve) or in the presence of MSCs (MSC/T-cell ratio of 1:10) (dashed curve). In some experiments, neutralizing monoclonal anti–MMP-2 or –MMP-9 antibodies were added (gray shaded). Neutralizing antibody was used to block the activity of MT1-MMP. IgG1 was used as an isotype control. T-cells were harvested at 72 h, stained with CD25-PE, and analyzed by flow cytometry. D: CD4+CD25− T-cells were activated by 10 μg/ml ConA for 24 h, washed three times to remove residual ConA, and resuspended in serum-free medium at final concentration of 2 × 105/ml. MMP-2 and MMP-9 recombinant proteins were added at final concentration of 1 mg/ml. T-cells were incubated with MMP recombinant proteins for an additional 6 h. MMP-2 or MMP-9 treated cells (solid curve) and untreated cells (dotted curve) were analyzed for CD25 expression by flow cytometry. Histograms of CD25 expression on T-cells are representative of three independent experiments.
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Figure 5: MSC-dependent reduction in CD25 expression by T-cells is mediated by MMP-2 and MMP-9. A: Enzymatic activities of MMP-2 and MMP-9 in medium conditioned by MSCs were analyzed by zymography. Conditioned medium was electrophoresed under nonreducing conditions and without heating through a 8% SDS-PAGE containing 0.1% gelatin. Following electrophoresis, the gels were incubated overnight in developing buffer containing SB-3CT to inhibit MMP activity. Gels were stained with Coomassie Blue, and densitometric quantification of MMP-2 and MMP-9 was performed using Scion software. Data were obtained from three individual experiments, and inhibition of MMP-2 and MMP-9 was determined by setting the mock treatment as 100%. □, control; ■, SB-3CT. B: To block activities of MMP-2 and MMP-9 in the MSC/T-cell coculture, SB-3CT was added at a concentration of 6 μmol/l. Stimulated T-cells alone (black curve) or cocultured with MSCs (dashed curve) or MSCs treated with SB-3CT (gray curve) were examined for surface expression of CD25. C: Stimulated T-cells were either cultured alone (black curve) or in the presence of MSCs (MSC/T-cell ratio of 1:10) (dashed curve). In some experiments, neutralizing monoclonal anti–MMP-2 or –MMP-9 antibodies were added (gray shaded). Neutralizing antibody was used to block the activity of MT1-MMP. IgG1 was used as an isotype control. T-cells were harvested at 72 h, stained with CD25-PE, and analyzed by flow cytometry. D: CD4+CD25− T-cells were activated by 10 μg/ml ConA for 24 h, washed three times to remove residual ConA, and resuspended in serum-free medium at final concentration of 2 × 105/ml. MMP-2 and MMP-9 recombinant proteins were added at final concentration of 1 mg/ml. T-cells were incubated with MMP recombinant proteins for an additional 6 h. MMP-2 or MMP-9 treated cells (solid curve) and untreated cells (dotted curve) were analyzed for CD25 expression by flow cytometry. Histograms of CD25 expression on T-cells are representative of three independent experiments.

Mentions: MMPs are zinc-dependent enzymes that bind histidine-containing motifs in components of the extracellular matrix and play a crucial role in embryogenesis, wound healing, and tissue remodeling (28–30). It has been determined that MMP-2 and MMP-9 may also play a role in immunological evasion of cancer cells by clearing CD25 from T-cell surfaces (31). The data in Figs. 4B and C confirm that the MSCs used in the present study contain and secrete MMP-2 and MMP-9, and the data in Fig. 4A are consistent with a role for these enzymes in MSC-dependent inhibition of T-cell responses by cleavage of CD25 from the cell surface. To verify that in our hands inhibition of MMP-2 and MMP-9 results in a detectable inhibition of MMP-dependent proteolysis, conditioned medium from MSC cultures was separated by SDS polyacrylamide electrophoresis in a gel containing gelatin as an MMP substrate and analyzed by zymography (32). The resultant gel was then incubated overnight at 37°C and the position-specific gelatin content determined by densitometry. Addition of SB-3CT at a concentration of 6 μmol/l resulted in a 52.4 and 90.5% inhibition of MMP-2 and MMP-9, respectively (Fig. 5A). To determine whether MMP-2 and/or MMP-9 can influence the surface expression of CD25 (Fig. 2C), bead-stimulated T-cells were cultured with MSCs in the presence or absence of 6 μmol/l SB-3CT and CD25 expression evaluated by flow cytometry. As shown in Fig. 5B, the inhibitor partially reversed the loss of CD25, and although the effect was not complete, the fact that the same concentration of inhibitor caused a complete abrogation of MSC dependent T-cell inhibition (Fig. 4A) suggests that maximal levels of CD25 expression are not required for optimal responses to IL-2.


Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9.

Ding Y, Xu D, Feng G, Bushell A, Muschel RJ, Wood KJ - Diabetes (2009)

MSC-dependent reduction in CD25 expression by T-cells is mediated by MMP-2 and MMP-9. A: Enzymatic activities of MMP-2 and MMP-9 in medium conditioned by MSCs were analyzed by zymography. Conditioned medium was electrophoresed under nonreducing conditions and without heating through a 8% SDS-PAGE containing 0.1% gelatin. Following electrophoresis, the gels were incubated overnight in developing buffer containing SB-3CT to inhibit MMP activity. Gels were stained with Coomassie Blue, and densitometric quantification of MMP-2 and MMP-9 was performed using Scion software. Data were obtained from three individual experiments, and inhibition of MMP-2 and MMP-9 was determined by setting the mock treatment as 100%. □, control; ■, SB-3CT. B: To block activities of MMP-2 and MMP-9 in the MSC/T-cell coculture, SB-3CT was added at a concentration of 6 μmol/l. Stimulated T-cells alone (black curve) or cocultured with MSCs (dashed curve) or MSCs treated with SB-3CT (gray curve) were examined for surface expression of CD25. C: Stimulated T-cells were either cultured alone (black curve) or in the presence of MSCs (MSC/T-cell ratio of 1:10) (dashed curve). In some experiments, neutralizing monoclonal anti–MMP-2 or –MMP-9 antibodies were added (gray shaded). Neutralizing antibody was used to block the activity of MT1-MMP. IgG1 was used as an isotype control. T-cells were harvested at 72 h, stained with CD25-PE, and analyzed by flow cytometry. D: CD4+CD25− T-cells were activated by 10 μg/ml ConA for 24 h, washed three times to remove residual ConA, and resuspended in serum-free medium at final concentration of 2 × 105/ml. MMP-2 and MMP-9 recombinant proteins were added at final concentration of 1 mg/ml. T-cells were incubated with MMP recombinant proteins for an additional 6 h. MMP-2 or MMP-9 treated cells (solid curve) and untreated cells (dotted curve) were analyzed for CD25 expression by flow cytometry. Histograms of CD25 expression on T-cells are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 5: MSC-dependent reduction in CD25 expression by T-cells is mediated by MMP-2 and MMP-9. A: Enzymatic activities of MMP-2 and MMP-9 in medium conditioned by MSCs were analyzed by zymography. Conditioned medium was electrophoresed under nonreducing conditions and without heating through a 8% SDS-PAGE containing 0.1% gelatin. Following electrophoresis, the gels were incubated overnight in developing buffer containing SB-3CT to inhibit MMP activity. Gels were stained with Coomassie Blue, and densitometric quantification of MMP-2 and MMP-9 was performed using Scion software. Data were obtained from three individual experiments, and inhibition of MMP-2 and MMP-9 was determined by setting the mock treatment as 100%. □, control; ■, SB-3CT. B: To block activities of MMP-2 and MMP-9 in the MSC/T-cell coculture, SB-3CT was added at a concentration of 6 μmol/l. Stimulated T-cells alone (black curve) or cocultured with MSCs (dashed curve) or MSCs treated with SB-3CT (gray curve) were examined for surface expression of CD25. C: Stimulated T-cells were either cultured alone (black curve) or in the presence of MSCs (MSC/T-cell ratio of 1:10) (dashed curve). In some experiments, neutralizing monoclonal anti–MMP-2 or –MMP-9 antibodies were added (gray shaded). Neutralizing antibody was used to block the activity of MT1-MMP. IgG1 was used as an isotype control. T-cells were harvested at 72 h, stained with CD25-PE, and analyzed by flow cytometry. D: CD4+CD25− T-cells were activated by 10 μg/ml ConA for 24 h, washed three times to remove residual ConA, and resuspended in serum-free medium at final concentration of 2 × 105/ml. MMP-2 and MMP-9 recombinant proteins were added at final concentration of 1 mg/ml. T-cells were incubated with MMP recombinant proteins for an additional 6 h. MMP-2 or MMP-9 treated cells (solid curve) and untreated cells (dotted curve) were analyzed for CD25 expression by flow cytometry. Histograms of CD25 expression on T-cells are representative of three independent experiments.
Mentions: MMPs are zinc-dependent enzymes that bind histidine-containing motifs in components of the extracellular matrix and play a crucial role in embryogenesis, wound healing, and tissue remodeling (28–30). It has been determined that MMP-2 and MMP-9 may also play a role in immunological evasion of cancer cells by clearing CD25 from T-cell surfaces (31). The data in Figs. 4B and C confirm that the MSCs used in the present study contain and secrete MMP-2 and MMP-9, and the data in Fig. 4A are consistent with a role for these enzymes in MSC-dependent inhibition of T-cell responses by cleavage of CD25 from the cell surface. To verify that in our hands inhibition of MMP-2 and MMP-9 results in a detectable inhibition of MMP-dependent proteolysis, conditioned medium from MSC cultures was separated by SDS polyacrylamide electrophoresis in a gel containing gelatin as an MMP substrate and analyzed by zymography (32). The resultant gel was then incubated overnight at 37°C and the position-specific gelatin content determined by densitometry. Addition of SB-3CT at a concentration of 6 μmol/l resulted in a 52.4 and 90.5% inhibition of MMP-2 and MMP-9, respectively (Fig. 5A). To determine whether MMP-2 and/or MMP-9 can influence the surface expression of CD25 (Fig. 2C), bead-stimulated T-cells were cultured with MSCs in the presence or absence of 6 μmol/l SB-3CT and CD25 expression evaluated by flow cytometry. As shown in Fig. 5B, the inhibitor partially reversed the loss of CD25, and although the effect was not complete, the fact that the same concentration of inhibitor caused a complete abrogation of MSC dependent T-cell inhibition (Fig. 4A) suggests that maximal levels of CD25 expression are not required for optimal responses to IL-2.

Bottom Line: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells.Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Immunology Group, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK. yunchuan.ding@nds.ox.ac.uk

ABSTRACT

Objective: Mesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.

Research design and methods: We investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.

Results: Our results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.

Conclusions: We demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

Show MeSH
Related in: MedlinePlus