Evaluating sphingosine and its analogues as potential alternatives for aggressive lymphoma treatment.

BACKGROUND: Ceramide (Cer) and sphingosine (Sph) interfere with critical cellular functions relevant for cancer progression and cell survival. While Cer has already been investigated as a potential drug target for lymphoma treatment, information about the potency of sphingosine is scarce. The aim of this study therefore was to evaluate Sph and its synthetic stereoisomer L-threo-sphingosine (Lt-Sph) as potential treatment options for aggressive lymphomas. METHODS: Diffuse large B cell lymphoma (DLBCL) cell lines were incubated with Sph and Lt-Sph and consequently analysed by flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to triple-quadrupole mass spectrometry (LC/MS/MS), electron microscopy, and Western blot. RESULTS: Sph induced cell death and blocked cell growth independently of S1P receptors in different DLBCL cell lines. Three different modes of Sph-mediated cell death were observed: Apoptosis, autophagy, and protein kinase C (PKC) inhibition. Generation of pro-apoptotic Cer accounted only for a minor portion of the apoptotic rate. CONCLUSION: Sph and its analogues could evolve as alternative treatment options for aggressive lymphomas via PKC inhibition, apoptosis, and autophagy. These physiological responses induced by different intracellular signalling cascades (phosphorylation of JNK, PARP cleavage, LC3-II accumulation) identify Sph and analogues as potent cell death inducing agents.

Sphingosine-1-phosphate receptor 3 promotes recruitment of monocyte/macrophages in inflammation and atherosclerosis.

RATIONALE: The role of sphingosine-1-phosphate (S1P) and its receptors in the pathogenesis of atherosclerosis has not been investigated. OBJECTIVE: We hypothesized that the S1P receptor 3 (S1P(3)) plays a causal role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: We examined atherosclerotic lesion development in mice deficient for S1P(3) and apolipoprotein (Apo)E. Although S1P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S1P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S1P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Bone marrow-derived S1P(3)-deficient macrophages produced less MCP-1 in response to lipopolysaccharide stimulation. In vitro, S1P was chemotactic for wild-type but not S1P(3)-deficient peritoneal macrophages. In vivo, S1P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S1P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S1P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S1P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S1P(3) in the arterial injury response. CONCLUSIONS: Our data suggest that S1P(3) mediates the chemotactic effect of S1P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

Local inactivation of sphingosine 1-phosphate in lymph nodes induces lymphopenia.

Sphingosine 1-phosphate (S1P) initiates T and B cell exit from lymphoid tissues by activating the S1P(1) receptor on lymphocytes. To define the mechanistic details of this ligand-receptor interaction, the biological activity of the S1P-blocking Ab Sphingomab was investigated. Treatment of mice with Sphingomab resulted in blood B and T cell lymphopenia. Although Sphingomab blocked S1P(1)-mediated calcium flux and receptor downregulation by S1P in vitro, plasma from Sphingomab-treated mice demonstrated a 4-fold increase in S1P concentration and largely retained its stimulating activity on S1P receptors. Plasma-borne S1P was obviously not sufficiently inactivated by Sphingomab to account for the observed lymphopenia. Therefore, we addressed the local S1P-blocking activity of Sphingomab in spleen and peripheral lymph nodes (pLNs) as a potential cause of PBL depletion. Transwell chemotaxis assays revealed the migration of freshly isolated splenocytes, but not pLN cells to S1P. However, chemotaxis of pLN cells was regained after culture in S1P-low medium, and pLN cells isolated from Sphingomab-treated mice also revealed enhanced chemotaxis to S1P, indicating substantial local inactivation of S1P in pLN after Sphingomab treatment. We conclude that treatment with the S1P-blocking Ab Sphingomab induces lymphopenia by inactivating S1P locally in pLN and not systemically in plasma. Consequently, the presence of local S1P amounts in secondary lymphoid organs contributes to B and T cell egress.

Redistribution of sphingosine 1-phosphate by sphingosine kinase 2 contributes to lymphopenia.

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2(-/-)) mice. In contrast to wild-type mice, SK2(-/-) mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2(-/-) mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2(-/-) mice were accompanied by higher S1P concentrations in blood, suggesting that SK2(-/-) mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2(-/-) mice, resulting in much higher C17-S1P concentrations in blood of SK2(-/-) mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.

Discontinued postnatal thymocyte development in sphingosine 1-phosphate-lyase-deficient mice.

Circulation of lymphocytes through peripheral lymphoid tissues as well as progenitor entry into the thymus and its output of mature T cells are critical for normal immune function. Egress of lymphocytes from both peripheral lymphoid organs and thymus is dependent on sphingosine 1-phosphate (S1P) gradients. S1P-lyase 1 (SGPL1) deficiency leads to accumulation of S1P in lymphoid tissues, which blocks lymphocyte egress and induces thymus atrophy. In this study, we investigated thymocyte development in SGPL1-deficient mice (SGPL1(-/-)), which exhibited postnatal discontinuation of early thymocytopoiesis starting at 2 wk after birth. SGPL(-/-) thymi showed a loss of developing thymocytes in the thymic cortex between 2 and 4 wk of age, whereas mature thymocytes accumulated in the medulla. Detailed analysis demonstrated a deficit in thymic early T cell progenitors (ETP) as the principal reason for discontinued thymocyte development. This developmental block was accompanied by accumulation of ceramides, resulting in enhanced apoptosis of developing T cells. Lack of immigration or settlement of ETP completely halted thymocyte development. We conclude that increased ceramide levels in the thymus of SGPL1(-/-) mice abrogate thymic development postnatally by enhanced thymocyte apoptosis and depletion of thymic ETP. Our findings indicate that potentially therapeutic immunosuppression by SGPL1 inhibition should benefit from monitoring ceramides to prevent their increase to apoptosis- inducing levels.

Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S1P from RBC into plasma is a regulated process that does not occur in plasma- or serum-free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non-redundant endogenous triggers for S1P release from RBC. S1P bound to SA and HDL was able to stimulate the S1P(1) receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S1P release, as demonstrated with the anti-S1P antibody Sphingomab. More S1P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S1P release in plasma. In co-cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S1P as a second function of RBC-associated S1P in the absence of SA and HDL and during tight RBC-EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S1P bound to SA and HDL, RBC-associated S1P was significantly incorporated by EC after S1P lyase (SGPL1) inhibition. RBC-associated S1P, therefore, has two functions: (1) It contributes to the cellular pool of SGPL1-sensitive S1P in tissues after transcellular transportation and (2) it helps maintain extracellular S1P levels via SA and HDL independently from SGPL1 activity.

Sphingosine 1-phosphate levels in plasma and HDL are altered in coronary artery disease.

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.

Accumulation of fingolimod (FTY720) in lymphoid tissues contributes to prolonged efficacy.

The immunomodulator fingolimod (FTY720) induces lymphopenia by inhibiting lymphocyte egress from thymus and secondary lymphoid organs (SLOs). It is phosphorylated mainly by sphingosine kinase (SK) 2 in vivo. FTY720-phosphate (FTY-P) activated and rapidly internalized S1P(1), which is the major sphingosine 1-phosphate (S1P) receptor for mediating lymphocyte egress. Although FTY-P is thought to be the active metabolite for triggering the onset of lymphopenia, nonphosphorylated FTY720 was much more potent in inhibiting cellular calcium flux and splenocyte chemotaxis via S1P(1) than FTY-P after preincubation. Determination of both compounds by liquid chromatography coupled to mass spectrometry revealed efficient uptake and accumulation of FTY720 but not FTY-P by splenocytes. Coculture experiments of B and T cells with and without FTY720 pretreatment led to rapid cellular transfer and phosphorylation by mouse lymphocytes. The presence of FTY720 in lymphoid tissues of FTY720-treated SK2-deficient mice without onset of lymphopenia excluded a potential role of the nonphosphorylated compound for lymphocyte egress. Local concentrations of both phosphorylated and nonphosphorylated FTY720 were much higher in lymphoid tissues than in blood. Therefore, we conclude that cellular accumulation of FTY720 generates a reservoir in thymus and SLOs, leading to sustained FTY-P production and activation of S1P(1) within tissues.

Glucocorticoids limit acute lung inflammation in concert with inflammatory stimuli by induction of SphK1.

Acute lung injury (ALI) is a severe inflammatory disease for which no specific treatment exists. As glucocorticoids have potent immunosuppressive effects, their application in ALI is currently being tested in clinical trials. However, the benefits of this type of regimen remain unclear. Here we identify a mechanism of glucocorticoid action that challenges the long-standing dogma of cytokine repression by the glucocorticoid receptor. Contrarily, synergistic gene induction of sphingosine kinase 1 (SphK1) by glucocorticoids and pro-inflammatory stimuli via the glucocorticoid receptor in macrophages increases circulating sphingosine 1-phosphate levels, which proves essential for the inhibition of inflammation. Chemical or genetic inhibition of SphK1 abrogates the therapeutic effects of glucocorticoids. Inflammatory p38 MAPK- and mitogen- and stress-activated protein kinase 1 (MSK1)-dependent pathways cooperate with glucocorticoids to upregulate SphK1 expression. Our findings support a critical role for SphK1 induction in the suppression of lung inflammation by glucocorticoids, and therefore provide rationales for effective anti-inflammatory therapies.

Immune regulation by sphingosine 1-phosphate and its receptors.

It is well established that the lysophospholipid and signalling molecule sphingosine 1-phosphate (S1P) has many important functions in immune surveillance. S1P is produced from sphingosine by two distinct sphingosine kinases, SphK1 and SphK2, and acts as an intracellular messenger and as an extracellular ligand of five G protein-coupled cell surface receptors designated S1P(1)-S1P(5). S1P not only regulates peripheral lymphocyte circulation, but also influences their differentiation, activation, infiltration, and local positioning. The therapeutic value of modulating S1P metabolism and S1P receptor function is currently tested in clinical trials and holds great promise for treatment of different autoimmune diseases. Despite its obvious contribution to immune regulation, the analysis of S1P is still challenging. A major obstacle is the difficulty to analyze S1P locally in tissues and within cells due to its high metabolic turnover and the limited resolution of current analytical techniques like liquid chromatography and mass spectrometry. This review focuses on recent advancements to our understanding how different sources of S1P contribute to immune function, and how changes in production, secretion, and degradation of S1P can influence immune responses.

Quantification of sphingosine-1-phosphate and related sphingolipids by liquid chromatography coupled to tandem mass spectrometry.

Liquid chromatography coupled to tandem mass spectrometry has evolved as the method of choice for the detection of sphingolipid metabolites due to its high sensitivity and superior specificity compared to other methodological approaches. Here, we describe a simple and robust method for the detection and quantification of sphingosine-1-phosphate (S1P) and related sphingolipids in biological samples. Tissue homogenates, cells, supernatant, plasma, and whole blood are spiked with an internal standard to account for loss of material during sample handling. After chloroform extraction of lipids under acidified conditions, the solvent is evaporated, and the remaining lipid extracts are dissolved in 20% CHCl(3) and 80% methanol. Following reversed-phase high-performance liquid chromatography step-gradient separation of sphingolipids and positive electrospray ionization, detection is carried out with the AB Sciex QTrap triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Characteristic fragment ions of S1P and related sphingolipids are monitored and subsequently analyzed relative to known standard concentrations of the pure compounds. Known problems of S1P quantification, such as carryover and insufficient HPLC separation, are discussed.