Elsevier

Cytokine

Volume 33, Issue 4, 21 February 2006, Pages 188-198
Cytokine

Tumor-derived macrophage migration inhibitory factor (MIF) inhibits T lymphocyte activation

https://doi.org/10.1016/j.cyto.2006.01.006Get rights and content

Abstract

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine that is considered a pro-inflammatory cytokine. However, our studies show that MIF, when produced in super-physiological levels by a murine neuroblastoma cell line (Neuro-2a) exceeding those normally seen during an immune response, inhibits cytokine-, CD3-, and allo-induced T-cell activation. MIF is also able to inhibit T cells that have already received an activation signal. The T-cell inhibitory effects of culture supernatants from neuroblastoma cells were reversed when the cells were transfected with dicer-generated si-RNA to MIF. When T cells were activated in vitro by co-culture with interleukin (IL)-2 and IL-15 and analyzed for cytokine production in the presence or absence of MIF-containing culture supernatant, inhibition of T-cell proliferation and induced cell death were observed even as the treated T cells produced high levels of interferon-gamma (IFN-γ). The inhibitory effects of MIF were partially reversed when lymphocytes from IFN-γ knockout mice were tested. We propose that the high levels of MIF produced by neuroblastoma cause activation induced T-cell death through an IFN-γ pathway and may eliminate activated T cells from the tumor microenvironment and thus contribute to escape from immune surveillance.

Introduction

Macrophage migration inhibitory factor (MIF) was first described as an activator of macrophage function that inhibits random macrophage migration. MIF is a 12.5 kDa protein secreted by macrophages, T cells, and other tissues, notably the anterior pituitary, that induces both gram-negative septic shock and delayed-type hypersensitivity reactions [1], [2], [3], [4]. MIF is unique in that the protein sequence and structural studies have not identified any cytokine family within which MIF can be placed. MIF is the only cytokine that is upregulated by glucocorticoids, and thus MIF plays a role in regulating host global responses to infection as glucocorticoid hormones are released from the hypothalamus-pituitary-adrenal-axis [3], [5]. Although a naturally occurring substrate has not been identified, MIF also has enzymatic properties and exhibits tautomerase and redox activity. This structural aspect of MIF is somewhat reminiscent of cyclophilin, which functions intracellularly as a peptidy-prolyl cis-trans isomerase; and can be secreted in response to proinflammatory stimuli [6], [7]. Studies have shown that MIF can override glucocorticoid inhibition of T-cell activation [8]. Furthermore, studies with a neutralizing anti-MIF antibody have shown that depletion of MIF from activated T-cell cultures inhibits T-cell proliferation in vivo and can inhibit the development of DTH lesions [8], [9].

Malignant tissues are able to blunt or alter anti-tumor immune responses by the production of soluble factors that act directly on lymphocytes [10], [11]. In our studies of the immune response to neuroblastoma, we found that both murine neuroblastoma cell lines and low passage clinical tumor isolates from either surgical resection of primary disease or bone marrow metastases produce MIF [12]. The list of other cytokines produced by neuroblastoma that could potentially influence the anti-tumor immune response also includes tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and IL-15 [13], [14], [15], [16]. The prevalence of each of these factors remains to be established, as in our limited analysis of clinical isolates only 1 in 5 produced IL-6, while 4 of 5 produced MIF. MIF has pleiotropic effects on both tumor cells and lymphocytes. MIF derived from neuroblastoma displays the classical functional activity of decreasing macrophage migration from glass capillary tubes, while also increasing transwell migration of tumor [12]. Studies with either tumor-derived, a parasite-encoded homolog, or recombinantly expressed MIF demonstrated that MIF not only inhibits macrophage random migration, but that it also plays a role in directional movement of tumor cells and macrophages [12], [17], [18].

The production of MIF by tumors appears contrary to its reported immuno-stimulatory activity. However, MIF plays a broad role in growth regulation in a number of systems. MIF is able to antagonize p53-mediated gene activation and apoptosis, and benzo[a]pyrene-induced fibrosarcomas in MIF knockout mice were smaller in size and had a lower mitotic index [19]. The MIF receptor has not been identified and is still under active investigation. CD74, the cell surface form of major histocompatibility class II-associated invariant chain, has been demonstrated to bind MIF with high affinity and is required for MIF-mediated extracellular signal-regulated kinase (ERK)-1/2 phosphorylation and PGE2 production in defined systems [20]. MIF has also been shown to have intracellular function in the regulation of cell growth via Jab1, a co-activator of AP-1 transcription [21].

With regard to the immune system, MIF is considered a broad-spectrum proinflammatory cytokine. MIF has been shown to stimulate TNF-α synthesis, is detected in DTH-reactive cells, and has been found to stimulate cognate T-cell activation [3], [22]. T cells activated by specific antigen, mitogen, or anti-CD3 antibody increase MIF mRNA expression and secretion of protein. Anti-MIF antibodies inhibit T-cell proliferation and B cell antibody production in vivo [8]. However, some studies have demonstrated negative effects of MIF on T-cell immunity. EG7 tumor-bearing mice treated with anti-MIF antibody show an increase in CD4 and CD8 T-cell accumulation at the tumor site and an increase in T-cell accumulation of adoptively transferred T cells [23]. Thus, decreasing MIF augmented the immune response to EG7. In studies described here, we sought to determine if tumor-encoded MIF has the potential to impact anti-tumor immunity. We found that high levels of tumor-derived MIF inhibited T-cell activation by preventing cell cycle progression and augmenting apoptosis through a specific signaling pathway that involved T lymphocyte-secreted interferon-gamma (IFN-γ). We speculate that the high levels of MIF produced by tumor cells results in activation-induced cell death of tumor-infiltrating T lymphocytes, thereby suppressing anti-tumor immunity.

Section snippets

Mice, tumor cells and reagents

A/J, C57BL/6 (termed B6) mice and IFN-γ knockout mice (B6.129S7-Ifngtm1Ts/J) were purchased from The Jackson Laboratory (Bar Harbor, MA) and used at 4–6 weeks of age. Mice were housed in the Biomedical Research Center at the Medical College of Wisconsin according to institutional guidelines. AGN2a is an aggressive subclone of the mouse Neuro-2a neuroblastoma cell line generated by serial in vivo passage [24]. Permanent cell lines transfected to express CD80 and CD137L (AGN2a/CD80-137L) were

In vitro demonstration of MIF knockdown

In previous work we described the production of soluble MIF in low passage cultures of patient-derived tumors and in a mouse neuroblastoma cell line, AGN2a [12]. To determine the functional consequences of inhibiting MIF production by AGN2a, endogenous MIF RNA was blocked with d-siRNA (dicer-generated small interfering RNA). d-siRNA was transfected into wild-type AGN2a, or an AGN2a-derived tumor vaccine cell line, AGN2a/CD80-137L (AGN2a permanently transfected with CMV-based mammalian

Discussion

During our study of the in vitro immune response to neuroblastoma we found that murine neuroblastoma cell lines inhibited rather than stimulated T lymphocyte activation in vitro. Unexpectedly, we determined that MIF is in large part the responsible agent. The MIF produced by neuroblastoma is secreted at super-physiological levels, exceeding 100 ng/ml in 3-day cultures of AGN2a. To date, MIF was thought to promote T-cell immunity as antibody depletion studies showed a critical requirement for MIF

Acknowledgments

We would like to thank Natalia Natalia for technical support, and acknowledge support from the Midwest Athletes Against Childhood Cancer (MACC Fund, Inc.) and from National Institutes of Health, NCI grant CA100030.

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