Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells

Abstract

The ability of mesenchymal stem cells (MSC) to suppress alloresponsiveness is poorly understood. Herein, an allogeneic mixed lymphocyte response was used as a model to investigate the mechanisms of MSC-mediated immunomodulation. Human MSC are demonstrated to express the immunosuppressive cytokines hepatocyte growth factor (HGF), interleukin (IL)-10 and transforming growth factor (TGF)-beta1 at concentrations that suppress alloresponses in vitro. MSC also express cyclooxygenase 1 and 2 and produce prostaglandin E2 constitutively. Blocking studies with indomethacin confirmed that prostaglandins contribute to MSC-mediated allosuppression. The proinflammatory cytokine interferon (IFN)-gamma did not ablate MSC inhibition of alloantigen-driven proliferation but up-regulated HGF and TGF-beta1. IFN-gamma also induced expression of indoleamine 2,3, dioxygenase (IDO), involved in tryptophan catabolism. Use of an antagonist, 1-methyl-L-tryptophan, restored alloresponsiveness and confirmed an IDO contribution to IFN-gamma-induced immunomodulation by MSC. Addition of the tryptophan catabolite kynurenine to mixed lymphocyte reactions (MLR), blocked alloproliferation. These findings support a model where IDO exerts its effect through the local accumulation of tryptophan metabolites rather than through tryptophan depletion. Taken together, these data demonstrate that soluble factors, or products derived from MSC, modulate immune responses and suggest that MSC create an immunosuppressive microenvironment capable of modulating alloresponsiveness even in the presence of IFN-gamma.

Figures

Fig. 1

Mesenchymal stem cells (MSC) suppress alloantigen-driven proliferation. MSC were co-cultured with peripheral blood mononuclear cells (PBMC) from major histocompatibility complex (MHC) mismatched donors (P1 or P2) for 96 h and alloantigen-driven proliferation measured by [3H]-thymidine incorporation expressed as mean counts per minute (cpm) ± standard error. Significantly reduced proliferation by responder PBMC (*P = 0·012) was determined by paired t-test analysis. Conditioned media (MSC s/n) obtained from 24-h MSC cultures suppressed alloantigen-driven proliferation (*P = 0·027). MSC or PBMC cultured alone are shown for comparison. Results are representative of three independent experiments each performed in triplicate. No significant loss of viability or induction of apoptosis was detected in this or subsequent experiments.

Fig. 2

Mesenchymal stem cell (MSC) production of immunomodulatory mediators determined by reverse transcription–polymerase chain reaction (RT–PCR) (a–c) and enzyme immunoassay (EIA) (d–f). mRNA was isolated from 6-h cultures of unstimulated MSC and subjected to semiquantitive reverse transcription–polymerase chain reaction (RT–PCR) as detailed in Methods for hepatocyte growth factor (HGF) (a), interleukin (IL)-10 (b) and transforming growth factor (TGF)-β1 (c). As human results may show variability, MSC derived from three separate donors (a–c: MSC 1, 2, 3) are shown. Supernatants from parallel cultures were also examined at 24 h for HGF (d), IL-10 (e) and TGF-β1 (f) protein expression by EIA. – indicates mRNA (a–c) from 6-h epithelial (A549) non-cytokine-secreting cell cultures or 24-h culture supernatants (d–f) as negative controls. EIA results are representative of three independent experiments using MSC from donor 1. EIA determinations were each performed in triplicate and expressed as mean concentration ± standard error.

Fig. 3

Immunosuppressive cytokines reduced alloantigen-driven proliferation but could not be reversed by neutralizing these factors. (a) mixed lymphocyte reactions (MLR) were performed with major histocompatibility complex (MHC) mismatched peripheral blood mononuclear cells (PBMC) (P1 and P2) but not mesenchymal stem cells (MSC). Selected cultures also contained recombinant human hepatocyte growth factor (HGF) (20 ng/ml), interleukin (IL)-10 (200 pg/ml) or transforming growth factor (TGF)-β1 (500 pg/ml). Cytokine concentrations were selected as comparable to that observed in Fig. 2. HGF, IL-10 and TGF-β1 significantly decreased alloantigen-driven proliferation (*P = 0·04, P = 0·044 and P = 0·018, respectively). In blocking studies (b) neutralizing antibodies against these cytokines (anti-HGF, 0·1–0·3 µg/ml; anti-TGF-β1, 1–6 µg/ml; anti-IL-10, 1–6 µg/ml) alone or in combination were added to MLR from MHC mismatched donors as above. Blocking any one of these factors alone or in combination did not reverse the inhibitory effect of MSC. Proliferation by responder PBMC are expressed as mean counts per minute ± standard error. Results are representative of at least three independent experiments, each performed in triplicate.

Fig. 4

Human mesenchymal stem cells (MSC) modulate alloresponses through prostaglandin production. (a) Expression of cyclooxygenase (COX)-1 and COX-2 mRNA by MSC or control prostaglandin E (PGE) non-producing fibroblast (–) cells was determined by reverse transcription–polymerase chain reaction (RT–PCR). MSC derived from three separate donors (MSC 1, 2, 3) are shown. (b) Constitutive PGE2 was detected in supernatants sampled from 24-h cultures of MSC but antagonized by indomethacin, as determined by specific enzyme immunoassay (EIA). (c) MSC were co-cultured in mixed lymphocyte reactions (MLR) from major histocompatibility complex (MHC) mismatched donors (P1 or P2) as in Fig. 1, but additionally cultures were established containing either indomethacin (20 µM) or acetyl sialic acid (aspirin, 1 µM). PGE2 inhibition by either indomethacin or aspirin significantly restored alloantigen driven proliferation (*P = 0·015 and P = 0·0158, respectively). Proliferation by responder peripheral blood mononuclear cells (PBMC) are expressed as mean counts per minute ± standard error. Results are representative of at least three experiments, each performed in triplicate.

Fig. 5

Interferon (IFN)-γ does not prevent mesenchymal stem cells (MSC) suppression of alloresponsiveness. Mixed lymphocyte reactions (MLR) were performed in the presence or absence of MSC as described in the legend to Fig. 1. MSC significantly suppress alloantigen-driven proliferation (**P = 0·0031). Additionally selected wells contained interferon (IFN)-γ (200 ng/ml) for the duration of the cultures. A significant decrease in proliferation by responder peripheral blood mononuclear cells (PBMC) was observed even in the presence of IFN-γ (**P = 0·0051). Proliferation by responder PBMC are expressed as mean counts per minute ± standard error. Results are representative of three independent experiments, each performed in triplicate.

Fig. 6

Interferon (IFN)-γ increases expression of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 and induces indoleamine 2,3, dioxygenase (IDO). Supernatants from 24-h cultures of mesenchymal stem cells (MSC) in the absence or presence of IFN-γ (200 ng/ml) were also examined for HGF (a), interleukin (IL)-10 (b) and transforming growth factor (TGF)-β1 (c); 24-h cultures of unstimulated or IFN-γ-stimulated MSC were lysed and assayed by semiquantitive transcription–polymerase chain reaction (RT–PCR) for IDO mRNA expression (d) as detailed in Methods. IFN-γ significantly increased HGF (**P = 0·0097) and TGF-β1 (***P = 0·0008) and did not reduce IL-10 expression following stimulation (P = 0·5). Enzyme immunoassay (EIA) results are expressed as mean counts per minute ± standard error and are representative of at least three independent experiments, each performed in triplicate.

Fig. 7

Indoleamine 2,3, dioxygenase (IDO) plays a role in mesenchymal stem cells (MSC)-mediated suppression of allo-responsiveness. Mixed lymphocyte reactions (MLR) were performed with mismatched donors (P1 and P2), as described in the legend to Fig. 1. Additionally, selected wells were treated with the IDO antagonist, 1-methyl l-tryptophan (mLT) (10 µM) (a) or with MSC replaced by the tryptophan breakdown product kynurenine (100 µM) (b). mLT significantly restored proliferation (a; *P = 0·047); conversely, addition of exogenous kynurenine significantly reduced allo-driven proliferation (b; **P = 0·0084). Kynurenine also accumulated in MLR containing MSC (c). Kynurenine concentration in supernatant from MLR with or without MSC was determined by high performance liquid chromatography (HPLC) as described in Methods. Kynurenine was significantly increased in MLR performed in the presence of MSC (*P = 0·022), peripheral blood mononuclear cells (PBMC) and MSC cultures are shown for comparison. Results are representative of three independent experiments, expressed as mean ± standard error, each performed in triplicate.