Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics |
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Authors: | Shobha Thangada Kamal M. Khanna Victoria A. Blaho Myat Lin Oo Dong-Soon Im Caiying Guo Leo Lefrancois Timothy Hla |
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Affiliation: | 1.Center for Vascular Biology, Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, Cornell University, New York, NY 10065;2.Center for Vascular Biology, and 3.Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030;4.Janelia Farm Campus, Howard Hughes Medical Institute, Ashburn, VA 20147 |
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Abstract: | The sphingosine 1-phosphate receptor 1 (S1P1) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P1 internalization is necessary for this effect. We characterize a knockin mouse (S1p1rS5A/S5A) in which the C-terminal serine-rich S1P1 motif, which is important for S1P1 internalization but dispensable for S1P1 signaling, is mutated. T cells expressing the mutant S1P1 showed delayed S1P1 internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P1 agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P1 expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P1 on T cells is a primary determinant of lymphocyte egress kinetics in vivo.Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator that signals via five G protein–coupled receptors (GPCRs), regulates vascular maturation, permeability, and angiogenesis (Hla, 2004; Cyster, 2005). Recently, interest in the roles of S1P and its receptors in the immune system has been prompted in part by the identification of the immunomodulator FTY720 (Brinkmann et al., 2002; Mandala et al., 2002; Chiba, 2005), which upon phosphorylation by Sphk2 to FTY720-P (Sanchez et al., 2003; Zemann et al., 2006) acts as a strong agonist for four out of five S1P receptors (Brinkmann et al., 2004). FTY720 induces profound lymphopenia by inhibiting the egress of lymphocytes from the thymus, peripheral lymph nodes, and Peyer’s patches (Chiba, 2005). Indeed, it is now appreciated that S1P signaling modulates the trafficking of not only naive and central memory T cells, but also B cells, dendritic cells, NK cells, osteoclasts, and hematopoietic progenitor cells (Allende and Proia, 2002; Kabashima et al., 2006; Massberg et al., 2007; Schwab and Cyster, 2007; Walzer et al., 2007; Ledgerwood et al., 2008; Rivera et al., 2008; Sebzda et al., 2008; Ishii et al., 2009). These studies suggest that S1P regulates hematopoietic and immune cell trafficking under homeostatic and disease conditions; however, it is unclear precisely how S1P receptor signaling modulates cellular responses to egress cues.The mechanism of how S1P regulates T cell trafficking has been intensively investigated; T cell–specific deletion of S1p1r or hematopoietic reconstitution using S1p1r−/− fetal liver cells resulted in profound lymphopenia, suggesting that the T cell–intrinsic S1P receptor 1 (S1P1) is essential for their egress from the thymus and secondary lymph nodes (Allende et al., 2004; Matloubian et al., 2004). This observation, coupled with the finding that FTY720-P induces the loss of cell-surface S1P1 from lymphocytes in an irreversible manner (Gräler and Goetzl, 2004; Matloubian et al., 2004), suggests that functional antagonism of S1P1 in the lymphocyte compartment is essential for the inhibition of T cell egress.However, other studies have led to the proposal of an alternative mechanism by which S1P1 regulates lymphocyte egress. Immunofluorescence microscopy demonstrated high expression levels of S1P1 in endothelial cells, whereas staining of lymphocytes was weaker (Singer et al., 2005; Sinha et al., 2009). Moreover, administration of SEW2971, a selective S1P1 agonist, does not induce irreversible receptor loss from the cell surface but causes significant lymphopenia in vivo (Jo et al., 2005). Two-photon microscopy of explanted lymph nodes containing labeled lymphocytes suggested that S1P1 agonists may modulate barrier function and closure of vascular portals in the medulla, through which T cells egress into efferent lymphatics (Wei et al., 2005). Thus, this alternative proposal favors endothelial cells as the primary target cell type for S1P1 agonists to inhibit lymphocyte egress (Rosen et al., 2008).Close interactions between immune and vascular cells may underlie the ability of S1P1 to promote lymphocyte egress. In lymph node cortical sinuses, egress of T and B cells required S1P1-dependent transendothelial traverse (Grigorova et al., 2009; Sinha et al., 2009). Indeed, competing chemotactic signaling between the egress-promoting S1P–S1P1 system and the retention-promoting CXCL21–CCR7 chemokine receptor system of T cells appears to determine the rate and extent of their egress from secondary lymphoid organs (Pham et al., 2008). Whether S1P1 signaling in lymphocytes, endothelial compartments, or both is important in the process of egress is not known.S1P1 is a type I GPCR that is rapidly phosphorylated upon agonist stimulation. Although several protein kinases are involved in the phosphorylation of S1P1 (Lee et al., 2001), phosphorylation at the C-terminal domain is particularly relevant to receptor desensitization and internalization (Hla, 2001). Because FTY720-P is degraded less efficiently than S1P by S1P lyase and S1P phosphatases (Bandhuvula et al., 2005; Mechtcheriakova et al., 2007; Yamanaka et al., 2008), its ligation likely induces sustained receptor activation kinetics. Presumably, this underlies the FTY720-P–induced irreversible internalization and proteosomal degradation of S1P1 and resultant lymphopenia (Oo et al., 2007). The GRK-2 enzyme is capable of phosphorylating the serine-rich motif in the C-terminal tail of S1P1 (Watterson et al., 2002), and we recently demonstrated that mutation of the five serines in the C terminus of S1P1 to nonphosphorylatable alanines inhibited S1P- and FTY720-P–induced receptor internalization in transfected HEK293 cells (Oo et al., 2007). Although previous studies of GPCR signaling and chemotaxis have provided some insights into the role of internalization in these processes, the results appear to be receptor specific. For example, a CXCR4 superagonist induced greater chemotaxis than the native ligand stromal cell–derived factor–1α (SDF-1α) with no perceptible receptor internalization (Sachpatzidis et al., 2003). Conversely, mutations in the C terminus of CXCR2 resulted in defective receptor internalization concomitant with impaired chemotaxis (Sachpatzidis et al., 2003). In the case of S1P1, it is unknown whether internalization is required for lymphocyte egress and recirculation.To address the role of S1P1 internalization in the control of lymphocyte egress during homeostasis and FTY720 treatment, we developed a mouse model in which WT S1P1 is replaced by the internalization-deficient mutant (S5A-S1P1). We show that although T cell trafficking under homeostasis is unaltered, S1p1rS5A/S5A mice display kinetic resistance to lymphopenia induced by the S1P1 modulator (FTY720-P) or disruption of the S1P gradient. Adoptive transfer of S1p1rWT/WT and S1p1rS5A/S5A lymphocytes and S1P1 surface staining of lymph node endothelial cells demonstrate that the T cell S1P1, and not endothelial cell S1P1 expression, regulates the rate of lymphocyte egress in vivo. These data support a T cell–intrinsic model of S1P1 signaling in egress kinetics wherein the internalization of S1P1 is a crucial modulator of the cues for T cell migration. |
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