Sphingosine 1-phosphate receptor agonists attenuate relapsing–remitting experimental autoimmune encephalitis in SJL mice

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Abstract

FTY720 is a prodrug for FTY-phosphate, an agonist at four of the five known receptors for sphingosine-1-phosphate (S1P). We show that administration of either FTY720 or FTY-P to SJL mice with established relapsing–remitting experimental autoimmune encephalitis (EAE) results in a rapid and sustained improvement in their clinical status, and a reversal of changes in expression of mRNAs encoding some myelin proteins and inflammatory mediators. EAE produced by adoptively transferring lymph node cells from immunized mice to naı̈ve hosts is similarly ameliorated by FTY-P. Treatment with FTY-P is accompanied by a dose-responsive peripheral lymphopoenia.

Introduction

FTY720 is a structural analog of myriocin, a metabolite of the ascomycete fungus Isaria sinclairia, with some structural resemblance to sphingosine, an endogenous lysolipid. Sphingosine undergoes phosphorylation via sphingosine kinase leading to the formation of sphingosine-1-phosphate (S1P), the cognate ligand for the family of S1P receptors (S1PR). Activation of S1P receptors results in a plethora of physiological actions such as chemotaxis, cellular differentiation, survival and growth, and regulation of actin-based cytoskeletal reorganization which leads to cell adherence and cell shape changes Goetzl and An, 1998, Chun, 1999, Fukushima et al., 2001.

FTY720 is a novel immunosuppressive agent which is active in various animal models of graft rejection and autoimmune disease, including graft versus host disease, type 1 diabetes, and rheumatoid arthritis. Chiba et al., 1996, Suzuki et al., 1996a, Suzuki et al., 1996b, Suzuki et al., 1998, Masubuchi et al., 1996, Matsuura et al., 2000. It is currently under development as an immunosuppressive agent for transplantation. It was thought initially that the mechanism of action was through induction of apoptosis in T lymphocytes Nagahara et al., 2000, Suzuki et al., 1996a, Suzuki et al., 1996b, Suzuki et al., 1997, and one effect of in vivo treatment with FTY720 is a profound lymphopoenia in the peripheral blood, with lymphocyte cell counts falling to as low as 5–10% of control levels at therapeutic doses of the compound. However, therapeutic effects are achieved in rats at doses of <1 mg/kg, at which the plasma concentrations are about two orders of magnitude lower than those required to drive T cell apoptosis in vitro (Yanagawa et al., 1998). A further argument against a role of apoptosis in most in vivo situations is provided by the observation that adoptively transferred fluorescently labeled lymphocytes disappear from the peripheral circulation on FTY720 treatment but reappear when drug treatment is discontinued (Pinschewer et al., 2000). It now appears that at least one of the mechanisms by which FTY720 achieves its effects in vivo is by a sequestration of circulating lymphocytes in peripheral lymph nodes Pinschewer et al., 2000, Brinkmann et al., 2000, Brinkmann et al., 2001a, Brinkmann et al., 2001b, Mandala et al., 2002, Xie et al., 2003.

FTY720 is a substrate for sphingosine kinase-2 (Sanchez et al., 2003) and phosphorylation in vivo (Mandala et al., 2002) has been demonstrated. The resultant ester (FTY-P) has a structure similar to sphingosine-1-phosphate, which is the preferred ligand at a group of G protein coupled receptors with five known members (S1P receptors, S1PR). Mandala et al. (2002) showed that FTY720 itself has weak or no activity at any of these receptors but that the phosphate ester is an agonist with low nanomolar potency at four of the five receptors, and these observations were further substantiated by Brinkmann et al. (2002). A nonhydrolysable phosphonate analogue of FTY-P retained sufficient potency at the four S1P receptors to have in vivo efficacy in a lymphopoenia assay. FTY720, sphingosine-1-phosphate, and the phosphonate analogue of FTY-P all caused a rapid and reversible peripheral lymphopoenia in rats and mice, reaching a nadir at 4 h postinjection. The potency of compounds in this assay reflected their intrinsic affinities at the four receptors, indicating that one or more of these receptors is indeed the molecular target of these compounds (Mandala et al., 2002). Recent reports using selective agonists demonstrate that S1P1 is the target for lymphopenia Forrest et al., 2004, Sanna et al., 2004. Moreover, lymphocytes genetically deleted for S1P1 have thymic emigration and recirculation defects similar to that achieved with the receptor agonists, suggesting that S1P1 is required for egress (Matloubian et al., 2004) and that the agonists induce lymphopenia by downregulating S1P1 on lymphocytes (Graler and Goetzl, 2004).

Two papers have appeared indicating that FTY720 is active in rodent models of experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. Brinkmann et al. (2002) treated Wistar rats with FTY720 (0.3 mg/kg/day) from the day of induction of EAE (day 0) in Wistar rats. They showed that rats thus treated did not develop EAE in this monophasic model. In a different acute monophasic rat model, in which myelin basic protein is used as the immunogen in Lewis rats, Fujino et al. (2003) showed once again that dosing rats orally from day 0 almost completely suppressed the development of disease. This was associated with a marked reduction in the number of T lymphocytes infiltrating the spinal cord and a reduction in the levels of the TH1 cytokines IL-2, IL-6, and interferon gamma. While these data are impressive, the efficacy of FTY720 when given at the onset of clinical symptoms or on established EAE is unknown. In the present investigation, we have examined the efficacy of FTY720 and its phosphate ester on an established disease state in the relapsing–remitting EAE model in SJL mouse (SJL rr-EAE), a model which mimics several features of human MS.

EAE can be induced in SJL strain mice by immunizing them with a peptide fragment of the myelin protein, proteolipid protein (PLP), together with pertussis toxin (PTX) treatment (McRae et al., 1992). The resulting EAE shares many features with human multiple sclerosis. It is a chronic disease from which the mice never recover and has a relapsing–remitting pattern similar to that of the major form of human multiple sclerosis McRae et al., 1992, McRae et al., 1995. In addition to the infiltration of T lymphocytes into the brain and the spinal cord (and unlike the common rat models), these mice also exhibit both demyelination and also axonal damage Sobel et al., 1990, Marracci et al., 2002. Demyelination has for many decades been regarded as the hallmark of multiple sclerosis, but in recent years, the importance of the axonal damage and eventual neuron loss as the key processes underlying the relentless progression of the disease has been recognised Trapp et al., 1998, Trapp et al., 1999, Wujek et al., 2002. In addition to confirming these features of the SJL-rr model, we also show that in this EAE model, significant changes in the quantitative expression of mRNAs encoding both myelin-related proteins and mediators such as granulocyte-macrophage colony stimulating factor (GM-CSF) and inducible nitric oxide synthase occur.

In this study, we characterise the effects of FTY720 and its phosphorylated derivative on the clinical state, and levels of circulating lymphocytes in the SJL mouse rr-EAE model. Initiating dosing just prior to the onset of clinical signs delays and blunts the first phase of the disease as previously reported in the rat models. More significantly for the clinical setting, if treatment with these compounds is delayed until the peak of the first phase of disease, there is an immediate and rapid improvement in the clinical status of the animals which is maintained for as long as dosing is continued. Treatment with FTY-P is associated with a partial reversal in the gene expression changes seen in untreated animals with the disease. We also show that there is a correlation between the magnitude of clinical improvement and the levels of lymphopoenia achieved at any given dose of FTY-P, but this correlation is incomplete, indicating that nonselective S1P receptor agonism may exert its effects in this model by additional mechanisms. FTY-P is also effective in improving clinical status and preventing mortality in an adoptive transfer version of the disease.

Section snippets

SJL mouse

Female SJL mice (6–9 weeks old) were obtained from the Jackson laboratory (Bar Harbor, ME). They were housed in a 12-h light / dark cycle with access to food and water ad lib. All animal procedures were conducted in accordance with protocols approved by the local animal care committee.

Active immunization

EAE was induced following the methods of McRae et al. (1992). A 20-mer peptide based on the mouse proteolipid protein (PLP) sequence 139–151 (His–Ser–Leu–Gly–Lys–Trp–Leu–Gly–His–Pro–Asp–Lys–Phe (custom synthesized

EAE in SJL mice

Mice injected subcutaneously with a single dose of PLP emulsified in CFA, together with intraperitoneal doses of PTX on the first and third days, began to lose body weight and manifest clinical signs at about day 7. The decline in body weight and increase in clinical scores occurred rapidly, reaching their maxima at about day 14. At this point, mice had typically lost about 30–35% of their body weight, and mean clinical scores were usually about 3. In our hands, the subsequent evolution of the

Discussion

FTY720 has been reported previously to be effective in ameliorating several autoimmune diseases, including EAE Chiba et al., 1996, Suzuki et al., 1996a, Suzuki et al., 1998, Masubuchi et al., 1996, Matsuura et al., 2000 Two previous studies have examined the effect of immunomodulation with FTY on rat models of EAE. Brinkmann et al. (2002) reported a prophylactic effect of FTY720 on the development of EAE in Wistar rats when administered from the day of immunization (0.3 mg/kg daily). A similar

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