The sphingosine-1-phosphate receptor-1 antagonist, W146, causes early and short-lasting peripheral blood lymphopenia in mice

Abstract

Agonists of the sphingosine-1-phosphate (S1P) receptors, like fingolimod (FTY720), are a novel class of immunomodulators. Administration of these compounds prevents the egress of lymphocytes from primary and secondary lymphoid organs causing peripheral blood lymphopenia. Although it is well established that lymphopenia is mediated by S1P receptor type 1 (S1P1), the exact mechanism is still controversial. The most favored hypothesis states that S1P1 agonists cause internalization and loss of the cell surface receptor on lymphocytes, preventing them to respond to S1P. Hence, S1P1 agonists would behave in vivo as functional antagonists of the receptor. For this hypothesis to be valid, a true S1P1 antagonist should also induce lymphopenia. However, it has been reported that S1P1 antagonists fail to show this effect, arguing against the concept. Our study demonstrates that a S1P1 antagonist, W146, induces a significant but transient blood lymphopenia in mice and a parallel increase in CD4+ and CD8+ lymphocytes in lymph nodes. Treatment with W146 also causes the accumulation of mature T cells in the medulla of the thymus and moreover, it induces lung edema. We show that both the S1P1 antagonist and a S1P1 agonist cause lymphopenia in vivo in spite of their different effects on receptor expression in vitro. Although the antagonist purely blocks the receptor and the agonist causes its disappearance from the cell surface, the response to the endogenous ligand is prevented in both cases. Our results support the hypothesis that lymphopenia evoked by S1P1 agonists is due to functional antagonism of S1P1 in lymphocytes.

Introduction

The role of the bioactive lipid sphingosine-1-phosphate (S1P) in the egress of lymphocytes from secondary lymphoid organs during immunosurveillance is well established [1]. Numerous lines of evidence indicate that S1P1 is the S1P receptor subtype involved in this process [2], [3]. Naïve lymphocytes, which express S1P1, are believed to exit secondary lymphoid organs sensing the gradient of S1P that exists between lymph nodes, lymph and blood, where the highest concentration of S1P is found [4], [5].

A fundamental contribution to this field was the discovery of fingolimod (Gilenia®, Novartis) [6], which has recently been approved as the first oral disease-modifying agent for the treatment of multiple sclerosis. Fingolimod is phosphorylated by sphingosine kinase 2 and converted into fingolimod phosphate, an agonist of S1P1, S1P3, S1P4 and S1P5 receptors [7]. The excellent clinical efficacy reported for fingolimod is attributed, at least in part, to the induction of peripheral lymphopenia [8]. This prevents autoreactive cells from migrating to the central nervous system to destroy the myelin sheath [6]. After fingolimod, more selective S1P1 agonists have been synthesized, showing the same lymphopenic effect as fingolimod in vivo [9], [10]. In addition to its role in lymphopenia, S1P1 may also be involved in other effects reported with fingolimod in experimental models in rodents, such as a direct effect on astrocytes in the central nervous system [11] or in promoting the generation and function of regulatory T cells [12]. However, the contribution of these effects to human clinical efficacy is unknown.

Interestingly, however, as well as administration of synthetic S1P1 agonists, genetic deletion of S1P1 in hematopoietic cells also causes inhibition of lymphocyte egress [2], [3]. This apparent contradiction has been explained by the fact that agonists induce internalization and sometimes degradation of the S1P1 expressed on lymphocytes, making cells unresponsive to the S1P gradient. This concept is known as functional antagonism, and is supported by several experimental studies [13], [14]. An alternative explanation known as the “stromal gate” hypothesis, points to endothelium as the main cellular target of the S1P1 agonists-induced lymphopenia. According to this hypothesis, compounds would act as S1P1 agonists on endothelium and potentiate the effect of the endogenous S1P in enhancing the endothelial barrier function. This would lead to the closure of vascular portals in the medulla, preventing cells to migrate to efferent lymphatics [15].

For the hypothesis of functional antagonism to be valid, internalization of S1P1 by an agonist should evoke the same in vivo effect on lymphocytes as the blockade of S1P1 by an antagonist. Thus, a strong argument against this hypothesis is that systemic administration of the S1P1 antagonists, VPC44116 or W146, has been shown not to cause lymphopenia in vivo [16], [17]. In our work, we have expanded initial in vivo studies with one of these S1P1 antagonists, compound W146. Our study clarifies the so far reported lack of lymphopenia induced by this compound. Additional effects following the treatment of mice with the S1P1 antagonist are also explored. In the light of our findings, we compare the in vivo and in vitro effects of the antagonist with that of a S1P1 agonist.