Beyond a structural component: sphingolipids in immunology

Two major classes of lipids participating in signaling cascades in immune cells are known today. One comprises glycerol-based lipids with diacylglycerol as its most prominent member that mediates the activation of classical and novel protein kinase C molecules. The second group contains the sphingolipids, with the best-investigated representatives being sphingosine, sphingosine-1-phosphate, and ceramide. In the last years the latter two molecules have especially received considerable attention for their modulatory capacity in the course of an apoptotic response. Today it is clear that sphingolipids are ubiquitously distributed in all eukaryotic cells, especially in cellular membranes, where they were previously thought to fulfil an exclusively structural role. Recent findings, however, have demonstrated functions beyond this. Sphingolipid specific G-protein coupled receptors were identified and their role as intracellular second messengers has been further elucidated. In addition, glycosphingolipids, in particular, are enriched in certain membrane compartments, known as detergent resistant membranes. These serve as entry sites for several receptor-mediated signaling events by stabilizing receptor/kinase interactions, suggesting an involvement in the initiation of signaling cascades. Altogether, these findings have led to new insights into both the role of these lipids in signaling as well as the underlying pathology of several diseases with imbalances in the sphingolipid metabolism. The development of these disorders has mainly been attributed to the toxic potential of lysosphingolipids up to now. In addition, attempts have been made to develop compounds and drugs containing the sphingolipid backbone for influencing diseases associated with unwanted cell activation (e.g, cancer, inflammatory processes). These novel findings and developments are reviewed in the following.     

The role of sphingosine and ceramide kinases in inflammatory responses

The 1-phosphates of sphingosine and ceramide (S1P and C1P) have emerged as key representatives of a new group of lipid signalling molecules. S1P is known to act both as an extracellular mediator and as an intracellular 'second messenger,' while C1P currently is only known for its intracellular actions. Therefore, sphingosine and ceramide kinases, the enzymes involved in the generation of these lipid mediators, are now in the spotlight. This review summarizes current information on structure, localization, substrate specificity, activation, and binding partners of these kinases, and then focuses on discoveries in relation to immune cell regulation and inflammation, addressing in particular mast cell activation and degranulation, IL-12 signalling, prostaglandin biosynthesis, monocyte activation, and neutrophil priming.     

Targeting ceramide metabolism with a potent and specific ceramide kinase inhibitor

Ceramide kinase (CerK) produces the bioactive lipid ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of ceramide, resulting in decreased endogenous C1P levels. When combined with another ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling ceramide metabolism that may provide insight into CerK physiological function.     

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P1 receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.     

Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis

The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of sphingosine 1-phosphate (S1P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.     

Sphingolipids: second messengers, mediators and raft constituents in signaling

Recently, evidence has accumulated to show that sphingolipids exert an important function in signaling. These lipids serve as intracellular second messengers and as extracellular mediators. Furthermore, glycosylated sphingolipids are essential components of membrane rafts, which serve as platforms for the initiation of signaling cascades. Here, Eva Prieschl and Thomas Baumruker summarize current findings in leukocytes illustrating these different facets.     

Prevention of CD40-Triggered Dendritic Cell Maturation and Induction of T-Cell Hyporeactivity by Targeting of Janus Kinase 3

Pharmacological targeting of Janus kinase 3 (JAK3) has been employed successfully to control allograft rejection and graft-vs.-host disease (GVHD). Recent evidence suggests that in addition to its involvement in common-gamma chain (cgamma) signaling of cytokine receptors, JAK3 is also engaged in the CD40 signaling pathway of peripheral blood monocytes. In this study, we assessed the consequences of JAK3 inhibition during CD40-induced maturation of myeloid dendritic cells (DCs), and tested the impact thereof on the induction of T-cell alloreactivity. Dendritic cells triggering through CD40 induced JAK3 activity, the expression of costimulatory molecules, production of IL-12, and potent allogeneic stimulatory capacity. In contrast, JAK3 inhibition with the rationally designed JAK3 inhibitor WHI-P-154 prevented these effects arresting the DCs at an immature level. Interestingly, DCs exposed to the JAK3-inhibitor during CD40-ligation induced a state of hyporeactivity in alloreactive T cells that was reversible upon exogenous IL-2 supplementation to secondary cultures. These results suggest that immunosuppressive therapies targeting the tyrosine kinase JAK3 may also affect the function of myeloid cells. This property of JAK3 inhibitors therefore represents a further level of interference, which together with the well-established suppression of cgamma signaling could be responsible for their clinical efficacy.     

A new superfamily of lymphoid and melanoma cell proteins with extensive homology to Schistosoma mansoni antigen Sm23.

A novel cDNA clone termed R2 was isolated by subtractive hybridization of a cDNA library of phytohemagglutinin (PHA)/phorbol myristate acetate-stimulated Jurkat cells and by rescreening a cDNA library of PHA-stimulated peripheral blood lymphocytes. It hybridizes to a single mRNA species of about 2.2 kb, which is inducible in lymphoid cells and codes for a protein of 267 amino acids which contains four potential transmembrane domains. A computer-aided comparison showed strong homology to four other membrane proteins, the pan B cell marker CD37, the pan leukocyte marker CD53, the melanoma antigen ME491 and, surprisingly, the Schistosoma mansoni antigen Sm23. The four human proteins share a number of additional similarities in their overall structure. These include identical spacing of the transmembrane domains, similar hydrophobicity plots, possible N-linked glycosylation sites of similar number and position as well as similar distribution of the cysteine residues. The majority of these characteristics are still conserved in the evolutionary most distant member of this family, the Schistosoma mansoni antigen Sm23. Here we introduce this new protein superfamily and characterize the inducible, lymphoid-specific member R2.     

Normal neutrophil functions in sphingosine kinase type 1 and 2 knockout mice

Sphingosine kinase (SPHK) has been implicated as an important element in neutrophil responses to diverse stimulatory agents. To get more insight into the role of the type 1 and 2 isoforms of SPHK in neutrophil functions, we made use of the respective SPHK knockout mice. Neutrophils isolated from the bone marrow of these mice showed normal increase of intracellular Ca(2+) when stimulated in vitro by fMLP, platelet-activating factor, the anaphylatoxin C5a, or ATP, and normal migration towards fMLP and C5a. Also, recruitment of neutrophils into the peritoneum towards the chemokines KC and MIP-2 or to LPS, and into the peripheral blood after fMLP injection was similar in SPHK knockout strains and wild-type animals. An in vivo model of bacterial lung infection revealed an accelerated progression of disease in SPHK2 (but not SPHK1) knockout mice as compared to wild-type controls. However, effector functions of SPHK-deficient neutrophils, such as superoxide production, beta-glucuronidase release and their capacity to kill bacteria were unchanged as compared to wild-type cells. To conclude, the data derived from SPHK knockout mice do not support the hypothesis that any of the two lipid kinases plays a crucial role in signalling downstream of various neutrophil stimuli; SPHKs appear not to be essential for neutrophil recruitment and effector functions.