Limits...
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.

Show MeSH

Related in: MedlinePlus

Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4+ T cells in vitro by induction of nitric oxide and prostaglandin E2 (PGE2). (a) CD4+ T cells, in the presence of accessory cells, were stimulated with 3 μg/ml anti-CD3 for 48 hours. Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) levels in the supernatant of these cultures were analyzed by Bio-Plex protein array system. Bars represent averages of three values ± standard error of the mean. (b-d) Wild-type MSCs were stimulated with IL-17 (20 ng/mL) or IFN-γ (100 U/mL) or both for 48 hours. cDNA samples were prepared and subjected to quantitative polymerase chain reaction analysis. The relative quantity of target mRNA levels was normalized for 18S RNA. Relative levels of programmed death ligand-1 (PD-L1) (b), inducible nitric oxide (iNOS) (c), and cyclo-oxigenase-2 (COX-2) (d) are shown. Bars represent averages of two values. ND, not detectable. (e) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated number of mitomycin c-treated wild-type MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. Co-cultures were grown in the absence (control) or presence of 200 μM 1-methyl-DL-tryptophan, 10 μM indomethacin, or 10 μM GW274150. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875665&req=5

Figure 3: Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4+ T cells in vitro by induction of nitric oxide and prostaglandin E2 (PGE2). (a) CD4+ T cells, in the presence of accessory cells, were stimulated with 3 μg/ml anti-CD3 for 48 hours. Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) levels in the supernatant of these cultures were analyzed by Bio-Plex protein array system. Bars represent averages of three values ± standard error of the mean. (b-d) Wild-type MSCs were stimulated with IL-17 (20 ng/mL) or IFN-γ (100 U/mL) or both for 48 hours. cDNA samples were prepared and subjected to quantitative polymerase chain reaction analysis. The relative quantity of target mRNA levels was normalized for 18S RNA. Relative levels of programmed death ligand-1 (PD-L1) (b), inducible nitric oxide (iNOS) (c), and cyclo-oxigenase-2 (COX-2) (d) are shown. Bars represent averages of two values. ND, not detectable. (e) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated number of mitomycin c-treated wild-type MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. Co-cultures were grown in the absence (control) or presence of 200 μM 1-methyl-DL-tryptophan, 10 μM indomethacin, or 10 μM GW274150. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown.

Mentions: These data demonstrate the importance of IFN-γ signaling in MSCs to suppress T-cell proliferation. To investigate which molecules are involved in the suppression of proliferation, quantitative PCR was performed on IL-17- and IFN-γ-stimulated wild-type MSCs. These stimuli were chosen based on their upregulated expression in CD4+ T cells by stimulation with anti-CD3 antibodies (Figure 3a) and because these cytokines have been shown to synergistically induce the expression of iNOS [34] and IDO [35] in fibroblasts. The expression of iNOS, IDO, PD-L1, and COX-2, molecules involved in inhibition of T-cell proliferation and known to be induced by IFN-γ in MSCs [11], was analyzed. Unstimulated MSCs expressed no or low levels of these inhibitory factors (Figure 3b-d). Upon stimulation with IL-17 or IFN-γ alone, expression of PD-L1 (Figure 3b), iNOS (Figure 3c), and COX-2 (Figure 3d) was upregulated mildly. However, when IL-17 and IFN-γ were added simultaneously, expression levels of PD-L1, iNOS, and COX-2 (Figure 3b-d) were synergistically upregulated. IDO mRNA could not be detected in unstimulated or stimulated MSCs (data not shown). These data indicate that IFN-γ acts synergistically with IL-17 to upregulate expression of PD-L1, iNOS, and COX-2 in MSCs, making these molecules candidate mediators of T-cell inhibition. The involvement of iNOS and COX-2 in inhibition of T-cell proliferation was demonstrated by the addition of inhibitors of these enzymes - GW274150 and indomethacin [8,36], respectively - to the co-cultures. The addition of these inhibitors resulted in the abrogation of the inhibition of T-cell proliferation by wild-type MSCs (Figure 3e). The addition of the IDO inhibitor 1-methyl-DL-tryptophan (1-MT) did not affect the inhibition conferred by MSCs (Figure 3e).


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)

Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4+ T cells in vitro by induction of nitric oxide and prostaglandin E2 (PGE2). (a) CD4+ T cells, in the presence of accessory cells, were stimulated with 3 μg/ml anti-CD3 for 48 hours. Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) levels in the supernatant of these cultures were analyzed by Bio-Plex protein array system. Bars represent averages of three values ± standard error of the mean. (b-d) Wild-type MSCs were stimulated with IL-17 (20 ng/mL) or IFN-γ (100 U/mL) or both for 48 hours. cDNA samples were prepared and subjected to quantitative polymerase chain reaction analysis. The relative quantity of target mRNA levels was normalized for 18S RNA. Relative levels of programmed death ligand-1 (PD-L1) (b), inducible nitric oxide (iNOS) (c), and cyclo-oxigenase-2 (COX-2) (d) are shown. Bars represent averages of two values. ND, not detectable. (e) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated number of mitomycin c-treated wild-type MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. Co-cultures were grown in the absence (control) or presence of 200 μM 1-methyl-DL-tryptophan, 10 μM indomethacin, or 10 μM GW274150. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875665&req=5

Figure 3: Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4+ T cells in vitro by induction of nitric oxide and prostaglandin E2 (PGE2). (a) CD4+ T cells, in the presence of accessory cells, were stimulated with 3 μg/ml anti-CD3 for 48 hours. Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) levels in the supernatant of these cultures were analyzed by Bio-Plex protein array system. Bars represent averages of three values ± standard error of the mean. (b-d) Wild-type MSCs were stimulated with IL-17 (20 ng/mL) or IFN-γ (100 U/mL) or both for 48 hours. cDNA samples were prepared and subjected to quantitative polymerase chain reaction analysis. The relative quantity of target mRNA levels was normalized for 18S RNA. Relative levels of programmed death ligand-1 (PD-L1) (b), inducible nitric oxide (iNOS) (c), and cyclo-oxigenase-2 (COX-2) (d) are shown. Bars represent averages of two values. ND, not detectable. (e) CD4+ T cells (5 × 104 cells) and accessory cells (5 × 104 cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated number of mitomycin c-treated wild-type MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [3H]TdR. Co-cultures were grown in the absence (control) or presence of 200 μM 1-methyl-DL-tryptophan, 10 μM indomethacin, or 10 μM GW274150. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown.
Mentions: These data demonstrate the importance of IFN-γ signaling in MSCs to suppress T-cell proliferation. To investigate which molecules are involved in the suppression of proliferation, quantitative PCR was performed on IL-17- and IFN-γ-stimulated wild-type MSCs. These stimuli were chosen based on their upregulated expression in CD4+ T cells by stimulation with anti-CD3 antibodies (Figure 3a) and because these cytokines have been shown to synergistically induce the expression of iNOS [34] and IDO [35] in fibroblasts. The expression of iNOS, IDO, PD-L1, and COX-2, molecules involved in inhibition of T-cell proliferation and known to be induced by IFN-γ in MSCs [11], was analyzed. Unstimulated MSCs expressed no or low levels of these inhibitory factors (Figure 3b-d). Upon stimulation with IL-17 or IFN-γ alone, expression of PD-L1 (Figure 3b), iNOS (Figure 3c), and COX-2 (Figure 3d) was upregulated mildly. However, when IL-17 and IFN-γ were added simultaneously, expression levels of PD-L1, iNOS, and COX-2 (Figure 3b-d) were synergistically upregulated. IDO mRNA could not be detected in unstimulated or stimulated MSCs (data not shown). These data indicate that IFN-γ acts synergistically with IL-17 to upregulate expression of PD-L1, iNOS, and COX-2 in MSCs, making these molecules candidate mediators of T-cell inhibition. The involvement of iNOS and COX-2 in inhibition of T-cell proliferation was demonstrated by the addition of inhibitors of these enzymes - GW274150 and indomethacin [8,36], respectively - to the co-cultures. The addition of these inhibitors resulted in the abrogation of the inhibition of T-cell proliferation by wild-type MSCs (Figure 3e). The addition of the IDO inhibitor 1-methyl-DL-tryptophan (1-MT) did not affect the inhibition conferred by MSCs (Figure 3e).

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