Elsevier

Immunology Letters

Volume 110, Issue 2, 15 June 2007, Pages 91-100
Immunology Letters

IFN-γ and TNF-α differentially regulate immunomodulation by murine mesenchymal stem cells

https://doi.org/10.1016/j.imlet.2007.04.001Get rights and content

Abstract

Murine mesenchymal stem cells (MSC) have the ability to inhibit allogeneic immune responses. Two different mechanisms, either cell contact-dependent or independent, have been proposed to account for this immunosuppression. The focus of this study was to elucidate the involvement of soluble suppressive factors secreted by murine MSC in an inflammatory setting, and their role in MSC immunomodulation. In a non-inflammatory environment, bone marrow derived murine MSC constitutively expressed low levels of COX-2, PGE-2, TGF-β1 and HGF, but not IL-10, PD-1, PD-L1 or PD-L2. These MSC were able to significantly reduce alloantigen driven proliferation in mixed lymphocyte reactions as well as mitogen driven proliferation. The pro-inflammatory cytokines IFN-γ and TNF-α did not ablate MSC mediated immunosuppression. MSC expression of PGE-2, IDO and PD-L1 was differentially regulated by these cytokines. COX-2 and PGE-2 expression by MSC were upregulated by both IFN-γ and TNF-α, and using a biochemical inhibitor this was shown to have an essential, non-redundant role in modulating alloantigen-driven proliferation. However, the surface expression of PD-L1 was induced by IFN-γ but not TNF-α and similarly functional IDO expression was only induced by IFN-γ stimulation. Blocking studies using neutralising antibodies and biochemical antagonists revealed that while PD-L1 induction was not essential, IDO expression was a prerequisite for IFN-γ mediated MSC immunomodulation. These data demonstrate that murine MSC expression of immunomodulatory factors dramatically changes in a pro-inflammatory environment and that IFN-γ in particular has an important role in regulating MSC immunomodulatory factor expression.

Introduction

Mesenchymal stem cells (MSC) are multipotent adult progenitor cells which have the ability to differentiate into a number of lineages [1], [2], [3]. These cells are primarily found in the bone marrow but have also been isolated from other sites in the body [4]. Recent advances in the isolation, culture and differentiation of bone marrow derived MSC have highlighted the potential use of these cells in regenerative medicine. A number of studies have shown beneficial effects of therapeutic MSC delivery in vivo. Horwitz et al. showed engraftment of donor MSC and the formation of new bone in a case of osteogenesis imperfecta [5]. HLA identical MSC were shown to be safe and associated with a reversal of pathology in a study of metachromatic leukodystrophy [6]. Surprisingly, engraftment of allogeneic human MSC resulted in improved bone marrow stroma reconstitution in a case of severe aplastic anaemia [7]. Mouse models have also proved useful in the development of regenerative medicine therapies and for probing immunological mechanisms. For example, administration of murine MSC before the onset of disease prevented pathology in EAE, a model of multiple sclerosis [8]. Similarly, administration of MSC before the onset of lung damage induced by bleomycin, ameliorated fibrotic effects and decreased inflammation [9]. Recently, MSC have also been shown to be anti-tumorigenic in a mouse model of Kaposi's sarcoma by inhibiting AKT activity [10].

A major caveat in the application of MSC based regenerative medicine therapies concerns the potential immune-mediated rejection of allogeneic cells. Intriguingly a growing body of evidence suggests that mismatched MSC evade regular immune allorecognition [2], [11], [12], [13], [14] and can inhibit immune responses [11], [15], [16], [17], [18]. The mechanisms by which MSC mediate these immunosuppressive effects have not been fully elucidated, however both contact dependent and independent mechanisms have been proposed [18], [19], [20]. Krampera and colleagues have shown that the suppressive activity of MSC was abrogated when cells were physically separated by a transwell membrane [18]. Other studies have shown that MSC may interfere directly with T cell, or antigen presenting cell phenotype, causing these cells to adopt regulatory functions [17], [18], [21], [22], [24], [25]. MSC may interfere with ligand–receptor interactions required for T cell activation and proliferation. In contrast, other studies have proposed a role for soluble factors in MSC mediated immunosuppression using transwell cultures or MSC supernatant [20], [26], [27]. Several candidate cytokines have been implicated in MSC mediated suppression, including IL-10 and TGF-β1.

The therapeutic use of MSC may well require delivery to sites of inflammation and the potential possibility for immunomodulatory activity by MSC under these conditions is not clear. Previously, we have proposed that MSC mimic many of the immunomodulatory aspects of the fetal allograft [28]. The fetal allograft uses an array of soluble factors with immunomodulatory potential as well as contact dependent mechanisms to prevent fetal loss [29], [30]. To address this hypothesis, several soluble factors known to be involved in immunosuppression by the fetal allograft were examined for their role in MSC mediated immunosuppression under inflammatory and non-inflammatory conditions.

In this study, we report the constitutive expression of low levels of immunomodulatory factors: COX-2, PGE-2, TGF-β1 and HGF by MSC. We demonstrate that high level expression of PD-L1, IDO and PGE-2 by MSC are differentially regulated by IFN-γ and TNF-α. Using blocking studies, the role of these factors was examined to show that murine MSC retain immunomodulatory capability under inflammatory conditions and that IFN-γ or TNF-α upregulation of PGE-2 may be of particular importance in maintaining this effect. Overall this study supports the potential therapeutic use of allogeneic MSC and provides support for a rational mechanism by which MSC evade allogeneic rejection despite the presence of inflammatory cytokines.

Section snippets

Animals

Six- to eight-week-old female BALB/c and C3H/HeN mice (Harlan, UK) were used for experiments under the guidelines of the Irish Department of Health and the research ethics committee of the National University of Ireland Maynooth.

Isolation and culture of bone marrow derived mesenchymal stem cells

Murine MSC were isolated and expanded using an in-house modification of the method of Peister and colleagues [31]. Murine MSC were obtained from 8- to 10-week old female BALB/c mice (Harlan, Oxon, UK). Mice were sacrificed by cervical dislocation and the femur and tibia

Murine MSC constitutively express low levels of COX-2 and the immunomodulatory mediators PGE-2, TGF-β1, and HGF

Murine MSC have the ability to suppress alloreactivity. The specific mechanisms by which this occurs have not yet been elucidated. Conflicting data report that this inhibitory effect could be mediated through either cell contact or through soluble factors [18], [19], [20], [23]. Initially, this study sought to focus on and to characterise the immunomodulatory cytokines that murine MSC express. In line with previous studies, murine MSC did not stimulate but significantly inhibited alloantigen

Discussion

In order to clarify the utility of using mesenchymal stem for regenerative medicine, the soluble and surface immunomodulatory molecules expressed by MSC were characterised under regular and inflammatory conditions. MSC retained the ability to suppress alloantigen driven proliferation in MLR in the presence of IFN-γ and TNF-α, however these cytokines used different mechanisms to achieve this effect. Both IFN-γ and TNF-α upregulated COX-2 and PGE-2 expression and using indomethacin we show that

Acknowledgements

This work was supported by the Science Foundation Ireland Centres for Science Engineering and Technology (CSET) funding of the Regenerative Medicine Institute (REMEDI). Dr Shirley O’Dea is thanked for advice on molecular techniques.

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