Leukemia inhibitory factor modulates production of inflammatory mediators and myelin phagocytosis by macrophages

https://doi.org/10.1016/j.jneuroim.2008.07.015Get rights and content

Abstract

Leukemia inhibitory factor (LIF) promotes survival of glial cells and neurons during autoimmune and injury responses in the central nervous system (CNS). While various studies indicate that LIF also modulates ongoing inflammatory responses, data on underlying events are lacking. In this study we demonstrate that LIF modulates macrophage function. LIF inhibits the production of oxygen radicals and TNFalpha and stimulates myelin uptake by macrophages. These effects of LIF are accompanied by activation of the JAK/STAT3 signalling pathway. Our findings demonstrate that LIF has anti-inflammatory properties and enhances myelin clearance, implicating that LIF may be an important factor in CNS inflammatory disease.

Introduction

Leukemia inhibitory factor (LIF) belongs to the interleukin (IL)-6 family of neuropoietic cytokines that signal through the common gp130 receptor subunit. Binding of LIF to its receptor complex leads to the activation of various signal transduction pathways such as the JAK/STAT3, ERK1/2 and the PI3 kinase pathway (Dekanty et al., 2006, Paling et al., 2004, Slaets et al., 2008). LIF has pleiotropic effects and is involved in e.g. stem cell renewal (Bauer and Patterson 2006), lipid metabolism (Marshall et al., 1994, Nonogaki et al., 1996), and macrophage maturation (Tanigawa et al., 1995). In the central nervous system (CNS) LIF acts as a neurotrophic factor (Murphy et al., 1991). During injury and inflammatory responses in the CNS, LIF production is especially induced in astrocytes (Banner et al., 1997). Previous work from our group shows that LIF is produced by macrophages and T cells in inflammatory brain lesions of multiple sclerosis (MS) patients (Vanderlocht et al., 2006). In addition, we demonstrated that both LIF receptor subunits, gp130 and LIFR-β, are present on macrophages whereas the LIFR-complex is almost absent on other cells of the immune system (Vanderlocht et al., 2006). So far, controversial reports exist on the action of LIF in neuroinflammatory responses. Treatment of animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, with LIF reduces clinical symptoms. Furthermore, neutralization of LIF exacerbates disease pathology in this model, suggesting that endogenous LIF is an important factor in limiting damage induced by CNS inflammatory responses (Butzkueven et al., 2002, Butzkueven et al., 2006). It was demonstrated that LIF prevents oligodendrocyte apoptosis in in vitro culture systems as well as in EAE whereas no significant effects on the inflammatory response were observed (Azari et al., 2006, Butzkueven et al., 2002, Vanderlocht et al., 2006). In contrast, Linker et al recently demonstrated that EAE symptoms are milder in LIF deficient mice during the chronic stage of disease. This was accompanied by increased levels of immune cell infiltrates and demyelination in the CNS (Linker et al., 2008). In line with these findings, overexpression of LIF in the injured spinal cord worsens clinical outcome. LIF overexpression in the CNS induced enhanced activation and proliferation of macrophages and microglia (Kerr and Patterson 2004). Moreover, LIF is chemotactic for macrophages and in LIF deficient mice neuroinflammatory responses are attenuated (Sugiura et al., 2000). These findings indicate LIF may have proinflammatory effects, although the direct influence of LIF on macrophages has not been investigated in detail.

Macrophages are considered important effector cells in neuroinflammatory diseases such as MS (Huitinga et al., 1990). Macrophages have an important role in demyelination and contribute to disease severity by the production of inflammatory mediators such as cytokines, oxygen radicals and proteases (Hendriks et al., 2005). However, macrophages may also exert beneficial effect in the CNS by removal of myelin debris and secretion of trophic factors (Kerschensteiner et al., 1999, Kotter et al., 2001). To elucidate the direct effect of LIF on macrophage functions implicated in neuroinflammation, we examined the influence of LIF on proinflammatory mediator production and myelin phagocytosis by macrophages. Our data show that LIF inhibits the production of reactive oxygen species and TNFα while it increases myelin uptake. In addition, LIF is produced by activated macrophages, but during myelin phagocytosis LIF production is reduced. Together these data indicate that LIF modulates macrophage function and may thereby be an important regulator of CNS inflammatory diseases like MS.

Section snippets

Isolation of mouse peritoneal macrophages

Macrophages were isolated from the peritoneal cavity of C57bl/6 mice (Harlan, Horst, The Netherlands) either directly or after thioglycollate elicitation. Mice were injected intraperitoneally with 2.5 ml of 3% thioglycollate (Sigma-Aldrich, Bornem, Belgium). After 4 days, peritoneal cells were isolated by lavage after injection of 4 ml PBS 5 mM EDTA, counted and plated at 1 × 106/ml in RPMI-1640 (Invitrogen, Merelbeke, Belgium), 10% FCS (Hyclone Europe, Erembodegem, Belgium), 1% sodiumpyruvate,

LIF inhibits the production of proinflammatory mediators by macrophages

To elucidate whether LIF affects proinflammatory mediator production by macrophages, it was investigated whether LIF influences the secretion of ROS and TNFα by macrophages.

First, we evaluated the expression of LIFR-β and gp130 on mouse peritoneal macrophages by flow cytometry using specific antibodies. Mouse peritoneal macrophages express significant levels of both receptor subunits that are further increased after activation with LPS (Fig. 1).

Next, the production of TNFα and oxygen species by

Discussion

In this study we demonstrate that LIF is an immunomodulatory cytokine. LIF inhibits the production of proinflammatory mediators by macrophages and modulates myelin phagocytosis. These data are important for our understanding of the role of LIF in CNS inflammation. We show that LIF suppresses the secretion of TNFα and ROS by macrophages. TNFα is produced by macrophages in the CNS during inflammatory responses and is a strong inducer of oligodendrocyte apoptosis (Slaets et al., 2008, Vanderlocht

Acknowledgement

J.J.A.H. would like to acknowledge the FWO (Fonds Wetenschappelijk Onderzoek—Vlaanderen) for a postdoctoral fellowship.

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