Unraveling the complexities of sphingosine-1-phosphate function: the mast cell model

Abstract

Sphingosine-1-phosphate (S1P) is a lipid mediator involved in diverse biological processes, from vascular and neural development to the regulation of lymphocyte trafficking. Many of its functions are regulated by five widely expressed S1P G-protein-coupled receptors (S1P(1-5)). S1P is produced mostly intracellularly, thus, much of its potential as an autocrine and paracrine mediator depends on how, when, and where it is generated or secreted out of the cells. However, S1P can also have intracellular activity independent of its receptors, adding to the complexity of S1P function. The mast cell, a major effector cell during an allergic response, has proven instrumental towards understanding the complex regulation and function of S1P. Antigen (Ag) engagement of the IgE receptor in mast cells stimulates sphingosine kinases, which generate S1P and are involved in the activation of calcium fluxes critical for mast cell responses. In addition, mast cells secrete considerable amounts of S1P upon activation, thus affecting the surrounding tissues and recruiting inflammatory cells. Export of S1P is also involved in the autocrine transactivation of S1P receptors present in mast cells. The in vivo response of mast cells, however, is not strictly dependent on their ability to generate S1P, but they are also affected by changes in S1P in the environment previous to Ag challenge. This review will discuss the recent advances towards understanding the intricacies of S1P generation, secretion and regulation in mast cells. In addition, how S1P receptors are activated and their involvement in mast cell functions will also be covered, including new insights on the role of S1P in the mast cell-mediated allergic response of systemic anaphylaxis.

FIGURE 1. Intracellular regulation of ceramide, sphingosine and sphingosine-1-phosphate

A variety of cell receptor agonists and environmental stimuli sequentially and/or selectively activate sphingomyelinases (that cleave the phosphocholine group of sphingomyelin to yield ceramide), ceramidases (that cleave the fatty acid chain of ceramide to form SPH), and sphingosine kinases (that phosphorylate SPH in its primary hydroxyl group to form S1P). Consequently, the levels of ceramide, SPH, S1P, or a combination of these lipids are elevated in cells [10]. All of them are bioactive lipids that can activate or inhibit various signaling pathways by affecting key signaling proteins. The actions of ceramide and SPH in many cell systems oppose those of S1P. S1P can act as a second messenger inside cells. Although the exact targets are unknown, it has been reported to affect a variety of calcium channels. S1P can be exported outside cells by transporters and bind a family of GPCR coupled receptors (S1P_1-5) present at the plasma membrane. Some isoforms of sphingomyelinase, ceramidase, and sphingosine kinase can be secreted under certain conditions, and the activation of these secreted enzymes can generate sphingolipid metabolites in the extracellular environment. Enzymes involved in the degradation of S1P (S1P phosphatases and S1P lyase) are also critical for the fine-tuning of S1P levels inside and outside cells. For clarity, the generation of sphingolipid metabolites is depicted at the inner leaflet of the plasma membrane, probably the major active signaling pool, but other intracellular membrane locations are possible (see reviews for more details [2, 10, 107, 108]).

FIGURE 2. Schematic representation of the different compartments involved in the regulation and function of S1P in vivo

At the intracellular compartment, S1P is formed after stimulation by different agonists, and it signals as an intracellular mediator. Other enzymes involved in the metabolism of sphingolipids (see Figure 1) can alter its levels and the levels of other metabolites to shape the cellular response. At the membrane compartment, S1P can exit the cell via plasma membrane transporters and engage autocrine (via binding to S1P receptors in the same cell type) or paracrine loops (via binding to S1P receptors in other cell types). At the extracellular compartment, local tissue S1P levels, circulating S1P or blood-tissue gradients may affect the expression of S1P receptors in different cells and their function in proximal or distant cells. The S1P present in the extracellular environment originates from the export of intracellularly generated S1P. Alternatively, secreted sphingolipid enzymes (see Figure 1) or, potentially, autotaxin can generate S1P extracellularly. Various stimuli or changes in physiological conditions can indirectly change S1P levels in the extracellular environment (i.e regulation of S1P lyase or S1P phosphatases (SPP), activation of platelets or mast cells, etc), affecting other cells responses. Reciprocally, activation of S1PR may alter the composition of external stimuli in the physiological environment.

FIGURE 3. Activation and function of sphingosine kinases in murine bone-marrow derived mast cells

After FcεRI engagement, the membrane-localized Src Kinases Lyn and Fyn form complexes with SphKs. This localizes SphKs to the membrane and to lipid rafts, where their substrate, SPH, is enriched. Fyn is necessary also for the activation of SphK1 and SphK2. Activated Fyn provides both Gab2/PI3K-dependent and -independent signals that are key to full activation of SphK1 and SphK2, respectively. The dependence on PI3K can be related to a direct effect of PIP3 on SphKs, its importance in PLD activation and phosphatidic acid generation, or other downstream signaling partners. SphK activities, in turn, may enhance Fyn and Lyn activities. SphK2 is essential for FcεRI-induced calcium influx from the extracellular media, PKC activation and consequent NFκB activation and thus, it affects degranulation, arachidonic acid, leukotriens and cytokine production. Furthermore, S1P production is required for the transactivation of the receptors S1P₁ and S1P₂. Presence of S1P₂ is important for proper degranulation, and S1P₁ for the to the movement of mast cells towards an Ag gradient. In murine mast cells, both SphK1 and SphK2 are necessary for chemotaxis towards Ag. An ABCC1-type of transporter is involved in the secretion of S1P into the media and in the transactivation of S1P₁ but not S1P₂. Other transporters may also participate in the secretion of S1P. Mast cell secreted S1P can promote inflammation by activating and recruiting other immune cells involved in allergic and inflammatory responses.

FIGURE 4. In vivo S1P networks affect mast cell function and anaphylaxis

This figure depicts the mast cell in its physiological environment, and the partnership between extrinsic S1P generated by cells other than mast cells (black arrows; “extracellular compartment” in Figure 2), and intrinsic S1P or S1P generated by stimulated mast cells (blue arrows; corresponding to the “intracellular compartment” in Figure 2), in the regulation of mast cell responsiveness. Loss of SphK2 dramatically reduces the intracellular levels of S1P in mast cells, while loss of SphK1 has no effect. In contrast, deficiency in SphK1 results in reduced levels of S1P in circulation while loss of SphK2 increases the levels. Mast cells may be affected by those changes in different ways. It is possible that changes in S1P in the circulation have a domino effect on the interstitial levels of S1P in tissues, particularly on cells in the proximity of blood vessels where mast cells are present. Changes in those levels may directly affect the priming of S1P₂ in the mast cell, impacting on degranulation once the cells are activated. This possibility implies interference of extrinsic S1P with the autocrine loop of S1P₂ activation by intrinsic S1P. Other possibilities include an effect of extrinsic S1P on mast cell precursors in the blood stream or mast cells in tissues that will change their phenotypic outcome towards a more or less responsive phenotype. Constant exposure to higher or lower levels of S1P could also alter the phenotype of immune or non-immune cells inducing the generation of mediators that secondarily might influence the differentiation of mast cells. As a consequence of these direct or indirect effects of circulating S1P, mast cells numbers or their phenotypic outcome could be modified.