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Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis.

Schurgers E, Kelchtermans H, Mitera T, Geboes L, Matthys P - Arthritis Res. Ther. (2010)

Bottom Line: In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA.Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. evelien.schurgers@rega.kuleuven.be

ABSTRACT

Introduction: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes.

Methods: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC.

Results: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.

Conclusions: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

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Treatment with mesenchymal stem cells (MSCs) of wild-type or interferon-gamma receptor knockout (IFN-γR KO) origin does not influence the development of collagen-induced arthritis in DBA/1 mice. Mice were immunized on day 0 with collagen type II (CII) in complete Freund's adjuvant and injected intravenously with MSCs on day 16 and day 23. The mean arthritic score (a) and the cumulative incidence of arthritis (b) in DBA/1 mice treated with phosphate-buffered saline (PBS), wild-type MSCs, or IFN-γR KO MSCs are shown. Error bars represent standard error of the mean (SEM). (c) On day 46, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (d) Forty-two days after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left ear. Delayed-type hypersensitivity responses were measured as the percentage of swelling (100 × [(thickness of the right ear -- thickness of the left ear)/thickness of the left ear]) at the indicated times. Histograms indicate averages ± SEM. (e) On day 19 after immunization with CII in complete Freund's adjuvant, DBA/1 wild-type mice were injected intravenously with 1 × 106 wild-type MSCs, IFN-γR KO MSCs, or PBS, followed by an administration of 10 μg of anti-CD3 antibody on day 20. On day 21, in vivo T-cell proliferation was measured by detection of 5-ethynyl-2' -deoxyuridine (EdU) in the T-cell populations in the spleen and lymph nodes by fluorescence-activated cell sorting analysis. The percentages of EdU-positive cells in the CD4+ and CD8+ populations in the spleen and lymph nodes are shown. Histograms represent averages of four mice ± SEM.
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Figure 4: Treatment with mesenchymal stem cells (MSCs) of wild-type or interferon-gamma receptor knockout (IFN-γR KO) origin does not influence the development of collagen-induced arthritis in DBA/1 mice. Mice were immunized on day 0 with collagen type II (CII) in complete Freund's adjuvant and injected intravenously with MSCs on day 16 and day 23. The mean arthritic score (a) and the cumulative incidence of arthritis (b) in DBA/1 mice treated with phosphate-buffered saline (PBS), wild-type MSCs, or IFN-γR KO MSCs are shown. Error bars represent standard error of the mean (SEM). (c) On day 46, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (d) Forty-two days after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left ear. Delayed-type hypersensitivity responses were measured as the percentage of swelling (100 × [(thickness of the right ear -- thickness of the left ear)/thickness of the left ear]) at the indicated times. Histograms indicate averages ± SEM. (e) On day 19 after immunization with CII in complete Freund's adjuvant, DBA/1 wild-type mice were injected intravenously with 1 × 106 wild-type MSCs, IFN-γR KO MSCs, or PBS, followed by an administration of 10 μg of anti-CD3 antibody on day 20. On day 21, in vivo T-cell proliferation was measured by detection of 5-ethynyl-2' -deoxyuridine (EdU) in the T-cell populations in the spleen and lymph nodes by fluorescence-activated cell sorting analysis. The percentages of EdU-positive cells in the CD4+ and CD8+ populations in the spleen and lymph nodes are shown. Histograms represent averages of four mice ± SEM.

Mentions: To test the possible involvement of MSCs in CIA, DBA/1 mice were immunized with CII in CFA on day 0 and injected intravenously with wild-type or IFN-γR KO MSCs at different time points (Table 1). In a first experiment, day -1 was chosen for treatment with MSCs because experiments previously performed in our laboratory demonstrated that one single injection of CD4+CD25+ regulatory T (Treg) cells at day -1 significantly inhibited CIA [37]. In fact, in this experiment, a group of mice that received Treg cells were included. Injection of either wild-type or IFN-γR KO MSCs at day -1 did not affect the severity or incidence of arthritis, whereas injection of Treg cells did reduce the severity of CIA. In two subsequent experiments, we considered treating the mice at later time points, when inflammation was already ongoing. Thus, MSCs were administered at day 16 (experiment 2 in Table 1) or at day 16 and 23 post-immunization (experiment 3 in Table 1 and Figure 4). Treatment of the mice did not influence the disease severity or the incidence of arthritis development (Table 1 and Figure 4a, b) as compared with PBS-treated control animals. To verify whether the failure of MSCs to affect clinical scores of arthritis was also reflected in cellular and humoral autoimmune responses, DTH and total anti-CII IgG were analyzed. Anti-CII IgG titers were similar between MSC-treated and PBS-treated mice (Figure 4c). In addition, DTH responses, as evident from the percentage of swelling upon challenge with CII, were not different between MSC-treated and control animals (Figure 4d). T-cell proliferation was also measured in these mice by injection of 10 μg of anti-CD3 antibody. The results revealed no differences in CD4+ and CD8+ T-cell proliferation in spleens and lymph nodes when arthritic mice were injected intravenously with wild-type or IFN-γR KO MSCs (Figure 4e). However, since T-cell activation is a combination of proliferation and cytokine production, the sera of anti-CD3-injected and MSC-treated mice were analyzed for cytokines. The serum of mice was pooled per group and analyzed for the T-cell cytokines IL-2, IL-5, IL-6, IL-10, and IFN-γ. The injection of anti-CD3 antibody caused a profound increase in cytokine levels in the sera of these mice. Treatment with wild-type or IFN-γR KO MSCs, however, did not result in a decrease of IL-2, IL-5, and IL-10 but slightly decreased the levels of IL-6 and IFN-γ (data not shown).


Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis.

Schurgers E, Kelchtermans H, Mitera T, Geboes L, Matthys P - Arthritis Res. Ther. (2010)

Treatment with mesenchymal stem cells (MSCs) of wild-type or interferon-gamma receptor knockout (IFN-γR KO) origin does not influence the development of collagen-induced arthritis in DBA/1 mice. Mice were immunized on day 0 with collagen type II (CII) in complete Freund's adjuvant and injected intravenously with MSCs on day 16 and day 23. The mean arthritic score (a) and the cumulative incidence of arthritis (b) in DBA/1 mice treated with phosphate-buffered saline (PBS), wild-type MSCs, or IFN-γR KO MSCs are shown. Error bars represent standard error of the mean (SEM). (c) On day 46, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (d) Forty-two days after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left ear. Delayed-type hypersensitivity responses were measured as the percentage of swelling (100 × [(thickness of the right ear -- thickness of the left ear)/thickness of the left ear]) at the indicated times. Histograms indicate averages ± SEM. (e) On day 19 after immunization with CII in complete Freund's adjuvant, DBA/1 wild-type mice were injected intravenously with 1 × 106 wild-type MSCs, IFN-γR KO MSCs, or PBS, followed by an administration of 10 μg of anti-CD3 antibody on day 20. On day 21, in vivo T-cell proliferation was measured by detection of 5-ethynyl-2' -deoxyuridine (EdU) in the T-cell populations in the spleen and lymph nodes by fluorescence-activated cell sorting analysis. The percentages of EdU-positive cells in the CD4+ and CD8+ populations in the spleen and lymph nodes are shown. Histograms represent averages of four mice ± SEM.
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Related In: Results  -  Collection

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Figure 4: Treatment with mesenchymal stem cells (MSCs) of wild-type or interferon-gamma receptor knockout (IFN-γR KO) origin does not influence the development of collagen-induced arthritis in DBA/1 mice. Mice were immunized on day 0 with collagen type II (CII) in complete Freund's adjuvant and injected intravenously with MSCs on day 16 and day 23. The mean arthritic score (a) and the cumulative incidence of arthritis (b) in DBA/1 mice treated with phosphate-buffered saline (PBS), wild-type MSCs, or IFN-γR KO MSCs are shown. Error bars represent standard error of the mean (SEM). (c) On day 46, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (d) Forty-two days after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left ear. Delayed-type hypersensitivity responses were measured as the percentage of swelling (100 × [(thickness of the right ear -- thickness of the left ear)/thickness of the left ear]) at the indicated times. Histograms indicate averages ± SEM. (e) On day 19 after immunization with CII in complete Freund's adjuvant, DBA/1 wild-type mice were injected intravenously with 1 × 106 wild-type MSCs, IFN-γR KO MSCs, or PBS, followed by an administration of 10 μg of anti-CD3 antibody on day 20. On day 21, in vivo T-cell proliferation was measured by detection of 5-ethynyl-2' -deoxyuridine (EdU) in the T-cell populations in the spleen and lymph nodes by fluorescence-activated cell sorting analysis. The percentages of EdU-positive cells in the CD4+ and CD8+ populations in the spleen and lymph nodes are shown. Histograms represent averages of four mice ± SEM.
Mentions: To test the possible involvement of MSCs in CIA, DBA/1 mice were immunized with CII in CFA on day 0 and injected intravenously with wild-type or IFN-γR KO MSCs at different time points (Table 1). In a first experiment, day -1 was chosen for treatment with MSCs because experiments previously performed in our laboratory demonstrated that one single injection of CD4+CD25+ regulatory T (Treg) cells at day -1 significantly inhibited CIA [37]. In fact, in this experiment, a group of mice that received Treg cells were included. Injection of either wild-type or IFN-γR KO MSCs at day -1 did not affect the severity or incidence of arthritis, whereas injection of Treg cells did reduce the severity of CIA. In two subsequent experiments, we considered treating the mice at later time points, when inflammation was already ongoing. Thus, MSCs were administered at day 16 (experiment 2 in Table 1) or at day 16 and 23 post-immunization (experiment 3 in Table 1 and Figure 4). Treatment of the mice did not influence the disease severity or the incidence of arthritis development (Table 1 and Figure 4a, b) as compared with PBS-treated control animals. To verify whether the failure of MSCs to affect clinical scores of arthritis was also reflected in cellular and humoral autoimmune responses, DTH and total anti-CII IgG were analyzed. Anti-CII IgG titers were similar between MSC-treated and PBS-treated mice (Figure 4c). In addition, DTH responses, as evident from the percentage of swelling upon challenge with CII, were not different between MSC-treated and control animals (Figure 4d). T-cell proliferation was also measured in these mice by injection of 10 μg of anti-CD3 antibody. The results revealed no differences in CD4+ and CD8+ T-cell proliferation in spleens and lymph nodes when arthritic mice were injected intravenously with wild-type or IFN-γR KO MSCs (Figure 4e). However, since T-cell activation is a combination of proliferation and cytokine production, the sera of anti-CD3-injected and MSC-treated mice were analyzed for cytokines. The serum of mice was pooled per group and analyzed for the T-cell cytokines IL-2, IL-5, IL-6, IL-10, and IFN-γ. The injection of anti-CD3 antibody caused a profound increase in cytokine levels in the sera of these mice. Treatment with wild-type or IFN-γR KO MSCs, however, did not result in a decrease of IL-2, IL-5, and IL-10 but slightly decreased the levels of IL-6 and IFN-γ (data not shown).

Bottom Line: In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA.Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, Faculty of Medicine, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium. evelien.schurgers@rega.kuleuven.be

ABSTRACT

Introduction: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes.

Methods: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC.

Results: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.

Conclusions: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

Show MeSH
Related in: MedlinePlus