Limits...
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.

Show MeSH

Related in: MedlinePlus

MMP-2 and MMP-9 are involved in the immunosuppression by MSCs. A: CFSE-labeled CD4+CD25− T-cells stimulated with anti-CD3/CD28 were cultured for 72 h in the presence or absence of MSCs. Cell cultures were treated with 1 mmol/l 1-MT or 1 mmol/l l-NMM or 1 mmol/l SnPP or 6 μmol/l SB-3CT. Proliferation was accessed by dilution of CFSE fluorescent intensity. Data from three independent experiments each performed in triplicate are represented as means ± SD. □, control; ○, 1-MT; ▴, l-NMMA; ●, SnPP; *SB-3CT. B: MSCs were seeded on 13-mm glass coverslips and fixed, permeabilized, and blocked with serum and incubated with mouse anti–MMP-9 or anti–MMP-2 monoclonal antibodies. Binding was visualized using an Alexa Fluro 488 FITC-conjugated secondary, and cells were counterstained with Hoechst 33342. Sections were examined using a Leica epifluorescence microscope. C: To collect conditioned medium, MSCs were washed with PBS three times and resupplied with serum-free medium for 24 h. After harvest, the MSC-conditioned medium was spun at 1,500g to remove cellular debris, loaded on SDS-PAGE gels, and blotted with monoclonal anti–MMP-2 or –MMP-9 antibodies. Recombinant mouse MMP-2 and MMP-9 were used as markers. Data shown are representative of three independent experiments. A high-quality digital representation of this figure is available in the online issue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712800&req=5

Figure 4: MMP-2 and MMP-9 are involved in the immunosuppression by MSCs. A: CFSE-labeled CD4+CD25− T-cells stimulated with anti-CD3/CD28 were cultured for 72 h in the presence or absence of MSCs. Cell cultures were treated with 1 mmol/l 1-MT or 1 mmol/l l-NMM or 1 mmol/l SnPP or 6 μmol/l SB-3CT. Proliferation was accessed by dilution of CFSE fluorescent intensity. Data from three independent experiments each performed in triplicate are represented as means ± SD. □, control; ○, 1-MT; ▴, l-NMMA; ●, SnPP; *SB-3CT. B: MSCs were seeded on 13-mm glass coverslips and fixed, permeabilized, and blocked with serum and incubated with mouse anti–MMP-9 or anti–MMP-2 monoclonal antibodies. Binding was visualized using an Alexa Fluro 488 FITC-conjugated secondary, and cells were counterstained with Hoechst 33342. Sections were examined using a Leica epifluorescence microscope. C: To collect conditioned medium, MSCs were washed with PBS three times and resupplied with serum-free medium for 24 h. After harvest, the MSC-conditioned medium was spun at 1,500g to remove cellular debris, loaded on SDS-PAGE gels, and blotted with monoclonal anti–MMP-2 or –MMP-9 antibodies. Recombinant mouse MMP-2 and MMP-9 were used as markers. Data shown are representative of three independent experiments. A high-quality digital representation of this figure is available in the online issue.

Mentions: It has previously been suggested that the suppression of T-cell proliferation mediated by MSCs is dependent on IDO (27), NO (10), and HO-1 (11). To determine whether these molecules play a role in the current system, specific inhibitors were added to the cultures of T-cells stimulated in the presence of graded numbers of MSCs. The inhibitors were used at concentrations previously reported to compromise suppression by MSCs. Inhibition of IDO (1-MT) and HO-1 (SnPP) had no effect individually on MSC-mediated T-cell suppression (Fig. 4A). Addition ofl-NG-monomethyl arginine citrate (l-NMMA), an inhibitor of NO synthase, partially restored proliferation at an MSC:T-cell ratio of 1:10, indicating that the MSC effect is at least partially dependent on NO. Significantly, however, the inhibition of proliferation was completely reversed by addition of SB-3CT, a specific inhibitor of MMP-2 and MMP-9, implicating these enzymes as necessary and sufficient for the inhibitory activity of MSCs.


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)

MMP-2 and MMP-9 are involved in the immunosuppression by MSCs. A: CFSE-labeled CD4+CD25− T-cells stimulated with anti-CD3/CD28 were cultured for 72 h in the presence or absence of MSCs. Cell cultures were treated with 1 mmol/l 1-MT or 1 mmol/l l-NMM or 1 mmol/l SnPP or 6 μmol/l SB-3CT. Proliferation was accessed by dilution of CFSE fluorescent intensity. Data from three independent experiments each performed in triplicate are represented as means ± SD. □, control; ○, 1-MT; ▴, l-NMMA; ●, SnPP; *SB-3CT. B: MSCs were seeded on 13-mm glass coverslips and fixed, permeabilized, and blocked with serum and incubated with mouse anti–MMP-9 or anti–MMP-2 monoclonal antibodies. Binding was visualized using an Alexa Fluro 488 FITC-conjugated secondary, and cells were counterstained with Hoechst 33342. Sections were examined using a Leica epifluorescence microscope. C: To collect conditioned medium, MSCs were washed with PBS three times and resupplied with serum-free medium for 24 h. After harvest, the MSC-conditioned medium was spun at 1,500g to remove cellular debris, loaded on SDS-PAGE gels, and blotted with monoclonal anti–MMP-2 or –MMP-9 antibodies. Recombinant mouse MMP-2 and MMP-9 were used as markers. Data shown are representative of three independent experiments. A high-quality digital representation of this figure is available in the online issue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712800&req=5

Figure 4: MMP-2 and MMP-9 are involved in the immunosuppression by MSCs. A: CFSE-labeled CD4+CD25− T-cells stimulated with anti-CD3/CD28 were cultured for 72 h in the presence or absence of MSCs. Cell cultures were treated with 1 mmol/l 1-MT or 1 mmol/l l-NMM or 1 mmol/l SnPP or 6 μmol/l SB-3CT. Proliferation was accessed by dilution of CFSE fluorescent intensity. Data from three independent experiments each performed in triplicate are represented as means ± SD. □, control; ○, 1-MT; ▴, l-NMMA; ●, SnPP; *SB-3CT. B: MSCs were seeded on 13-mm glass coverslips and fixed, permeabilized, and blocked with serum and incubated with mouse anti–MMP-9 or anti–MMP-2 monoclonal antibodies. Binding was visualized using an Alexa Fluro 488 FITC-conjugated secondary, and cells were counterstained with Hoechst 33342. Sections were examined using a Leica epifluorescence microscope. C: To collect conditioned medium, MSCs were washed with PBS three times and resupplied with serum-free medium for 24 h. After harvest, the MSC-conditioned medium was spun at 1,500g to remove cellular debris, loaded on SDS-PAGE gels, and blotted with monoclonal anti–MMP-2 or –MMP-9 antibodies. Recombinant mouse MMP-2 and MMP-9 were used as markers. Data shown are representative of three independent experiments. A high-quality digital representation of this figure is available in the online issue.
Mentions: It has previously been suggested that the suppression of T-cell proliferation mediated by MSCs is dependent on IDO (27), NO (10), and HO-1 (11). To determine whether these molecules play a role in the current system, specific inhibitors were added to the cultures of T-cells stimulated in the presence of graded numbers of MSCs. The inhibitors were used at concentrations previously reported to compromise suppression by MSCs. Inhibition of IDO (1-MT) and HO-1 (SnPP) had no effect individually on MSC-mediated T-cell suppression (Fig. 4A). Addition ofl-NG-monomethyl arginine citrate (l-NMMA), an inhibitor of NO synthase, partially restored proliferation at an MSC:T-cell ratio of 1:10, indicating that the MSC effect is at least partially dependent on NO. Significantly, however, the inhibition of proliferation was completely reversed by addition of SB-3CT, a specific inhibitor of MMP-2 and MMP-9, implicating these enzymes as necessary and sufficient for the inhibitory activity of MSCs.

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