Polyclonal immunoglobulins (IVIg) modulate nitric oxide production and microglial functions in vitro via Fc receptors

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Abstract

Controlled trials in multiple sclerosis (MS) and case reports in acute demyelinating encephalomyelitis (ADEM) have shown that intravenous immunoglobulins (IVIg) are of therapeutic benefit in central nervous system (CNS) inflammatory diseases. Studies in experimental autoimmune encephalomyelitis (EAE) have suggested these effects are mediated by modulation of the cytokine network and T cell responses. However, there are no data on the influence of IVIg on the local immune reaction in the CNS, the site of inflammation in EAE. We have therefore studied the effect of IVIg on cultured rat microglia, the main immune cell in the CNS. IVIg increased nitric oxide (NO) production in a dose-dependent manner in microglia stimulated with IFNγ. The increase was only marginal in LPS-treated cells, and no effect was seen in untreated microglia or after stimulation with TNFα or PMA. This enhancement of NO production depended on the Fc portion of IVIg and could be abrogated by the pharmacological inhibition of Syk and phosphatidylinositol 3-kinase, two enzymes involved in the signalling cascade of Fc receptors. TNFα secretion was dose-dependently stimulated by IVIg in both untreated microglia and after stimulation with LPS or IFNγ. Again, this effect was mediated through the Fc portion. Finally, we showed that Fc receptor-mediated phagocytosis was inhibited by IVIg, presumably by blockade of the Fc receptor. These different effects may protect oligodendrocytes from antibody mediated phagocytosis and on the other hand could terminate the immune reaction by induction of apoptosis in infiltrating T cells via NO and TNFα. We propose that IVIg, in addition to known effects on the peripheral immune system, may also modulate the local immune reaction in CNS inflammatory disease.

Introduction

Intravenous immunoglobulins (IVIg) have become an established component of immunomodulatory therapy in neurological autoimmune disorders, including inflammatory diseases of the central nervous system (CNS) (van der Meché and van Doorn, 1997, Dalakas, 1999, Stangel et al., 1999). In multiple sclerosis (MS), IVIg were shown in controlled studies to reduce the relapse rate and the number of gadolinium enhancing lesions (Fazekas et al., 1997, Achiron et al., 1998, Sorensen et al., 1998), and case reports also suggest a beneficial effect in acute disseminated encephalomyelitis (ADEM) (Hahn et al., 1996, Pradhan et al., 1999). Several mechanisms of action have been suggested from both in vivo and in vitro investigations, including modulation of the cytokine network, complement inhibition, and modification of B cell, T cell and monocyte functions (Andersson et al., 1996, Mouthon et al., 1996, Stangel et al., 1999). In experimental autoimmune encephalomyelitis (EAE), an animal model of inflammatory CNS disease, efficacy of IVIg was shown, and it was proposed that the effect is mediated by suppression of TNFα secretion and T cell modulation (Achiron et al., 1994, Pashov et al., 1998). However, there is no information on the immunomodulatory capacity of IVIg in the CNS. Activation of microglia, the principal immunological cell in the CNS, plays an important role in CNS inflammatory diseases (Brück et al., 1995, Sriram and Rodriguez, 1997). Upon stimulation, microglia produce a vast number of immunomodulatory substances like cytokines or nitric oxide (NO) (Spranger and Fontana, 1996, González-Scarano and Baltuch, 1999).

NO is a small molecule synthesized from the substrate l-arginine by the enzymatic reaction of NO synthases (NOS). Its role in vivo as messenger involves various functions during physiological and pathological processes (Lowenstein and Snyder, 1992, Schmidt and Walter, 1994). NOS can also be found in MS lesions, suggesting that NO may play a role in MS pathology (Bo et al., 1994, De Groot et al., 1997). Initially, NO was thought to be mainly toxic, and damage of oligodendrocytes by microglial NO has been suggested (Merrill et al., 1993). However, studies examining the role of NOS blockade in EAE are not conclusive (Parkinson et al., 1997, Kolb and Kolb-Bachofen, 1998), and recent investigations have shown that NO may even have a protective, anti-apoptotic effect under certain conditions (Nicotera et al., 1997, Dimmeler and Zeiher, 1997) or modulate the immune response (Kolb and Kolb-Bachofen, 1998). NO production was demonstrated to be downregulated in endothelial cells treated with IVIg (Schussler et al., 1996). We have therefore assessed in vitro the modulation of microglial NO production.

Section snippets

Cell culture

Microglia were isolated from neonatal Sprague–Dawley rat cerebra by the method of Giulian and Baker as previously described (Giulian and Baker, 1986, Hall et al., 1997). Briefly, brains were carefully stripped of the meninges and dissociated using mechanical shearing and trypsin (Sigma, Poole, UK). Cells were plated at two brains per polylysine (Sigma) coated 75-cm2 culture flask (Iwaki, Bibby-Sterilin, Stone, UK) in DMEM (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS;

Effect of IVIg and controls on the NO production of unstimulated microglia

Microglia are known to produce NO upon a variety of stimuli (Murphy et al., 1993). Under our experimental conditions, lipopolysaccharide (LPS, 1 μg/ml) was the most potent and reliable stimulator after treatment for 24 h (see Table 1). To verify that the measured nitrite indeed derived from NO production, the NOS inhibitor l-nitro-l-arginine-methyl ester (l-NAME, 1 mM) was used to block LPS-induced nitrite production. Interferon-γ (IFNγ, 100 U/ml) and phorbol myristate acetate (PMA, 1 μg/ml)

Discussion

We show here that IVIg can modulate microglial functions in vitro. While NO production was only enhanced in IFNγ stimulated cells, TNFα secretion was induced by IVIg alone and increased in stimulated microglia. Both of these effects are mediated through the Fc portion of IVIg. In contrast, Fc receptor mediated phagocytosis is inhibited by IVIg, probably due to blockade of the receptor. These differential effects demonstrate that there is not a general activation of microglia by IVIg and that

Acknowledgements

The study was partially supported by a research scholarship from the Deutsche Forschungsgemeinschaft (Sta 518/1-1) to M.S., and used facilities in the MRC Cambridge Centre for Brain Repair and the Universitätsklinikum Benjamin Franklin, FU Berlin.

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