Sphingosine Kinase1 Is Pivotal for FcεRI-Mediated Mast Cell Signaling and Functional Responses In Vitro and In Vivo

Atopic allergy is characterized by elevated IgE Abs that, in the presence of an allergen, activate the high affinity IgE receptor (FcεRI) to release inflammatory mediators from mast cells. As mast cell degranulation is pivotal to allergic diseases, investigating novel pathways that trigger mast cell degranulation could have important therapeutic potential (1, 2, 3).

The strongest known physiological stimulus for mast cell activation is cross-linking of the high-affinity receptor for IgE (FcεRI), leading to exocytosis of granules containing preformed inflammatory mediators and the de novo synthesis of pharmacologically active eicosanoids and proinflammatory cytokines and chemokines (1, 2, 3). It is well established that these mediators lead to hypersensitivity responses, as well as the propagation of inflammation.

In the last few years, it has become clear that sphingolipids are sources of important signaling molecules. In particular, the sphingolipid metabolite, sphingosine-1-phosphate (S1P),³ has been implicated in a variety of cellular processes, such as cell differentiation, apoptosis, and proliferation (4, 5, 6, 7), as well as in the regulation of the immune responses (8). Interestingly, S1P levels are elevated in the bronchoalveolar lavage fluid of asthmatic patients after Ag challenge, suggesting that S1P is relevant in allergic responses (Ref. 9 , reviewed in Ref. 10).

We and others have shown that in human, rat, and mouse mast cells, sphingosine kinase1 (SphK1) is required for FcεRI to trigger intracellular signals and proinflammatory responses (11, 12, 13). Moreover, we recently showed that SphK1 is one of the earliest genes to be activated during IgE-mediated mast cell priming (14), which is a prerequisite for allergic diseases. In contrast, a recent study, using mast cells-derived from SphK1 or SphK2 knockout (KO) mice, suggests that SphK2 is the isoform used by FcεRI to mediate degranulation and cytokine and eicosanoids production (15). In contrast, a more recent study using small interfering RNA (siRNA) to silence the SPHK genes in human mast cells, shows that SphK1, but not SphK2, plays a critical role in Ag-induced degranulation migration and cytokine and eicosanoids production, with SphK2 playing a lesser role and only for the partial secretion of TNF-α (16). Thus, given the differences between silencing SPHK genes in wild-type cells and those cells where the kinases were deleted in the preimplantation embryo, it is possible that the differences observed are due to a compensatory mechanism during embryonic development of the KO mice and that SphK1 may indeed also be required for mouse mast cell degranulation and for the release of other proinflammatory mediators from murine mast cells in vitro and in vivo.

In this study we have specifically silenced SPHK1 or SPHK2 in vitro and in vivo, using specific siRNAs, and demonstrate that SphK1, but not SphK2, is indeed required for the effector responses triggered by Ag/FcεRI in murine mast cells. Moreover, silencing SPHK1 in vivo strongly inhibited mast cell IgE/Ag-mediated anaphylaxis, whereas silencing SPHK2 had no protective effect at all. Our data indicate that SphK1 is the major isoform responsible for triggering the release of proinflammatory mediators from mast cells in vitro and in vivo, and validates SphK1 as a potential novel therapeutic target for mast cell-mediated allergic/inflammatory responses. Moreover, our results have another potential impact: the observation that siRNA methodology can generate results different to those of gene-knockout methods, which has not been well-documented.

Murine mast cells were isolated from the peritoneal cavity and cultured as previously described (17). Another source of mast cells was derived from bone marrow: total bone marrow samples from femurs of mice were cultured in RPMI 1640 medium supplemented with 10% FCS, 10 ng/ml IL-3, 20 ng/ml SCF, 2 mM l-glutamine, 100 U/ml penicillin, and streptomycin in a 5% CO₂ atmosphere. After 8 wk, bone marrow-derived mast cells were collected and tested for c-kit and FcεRI expression by flow cytometry using anti-c-kit (BD Biosciences) and anti-FcεRI (eBioscience) Abs.

For the siRNA studies, cells were incubated for 48 h with 1 μg/ml siRNAs for the mSphK1 and mSPHK2, and mixed with Lipofectamine (Invitrogen) for transfection. siRNA sequence for the mSphK1 is: (GGGCAAGGCUCUGCAGCUCdTT and GAGCUGCAGAGCCUUGCCCdTT); siRNA-sequence for the mSphK2 is: (GGCUAAGAUCUAUCAUdTT and AUGAUAGAUCUUAGCCdTT).

Mast cells were sensitized with 1 μg/ml mouse DNP-specific IgE (Sigma-Aldrich) overnight. Then cells were collected, washed, resuspended in RPMI 1640–1% FBS, and activated with 1 μg/ml Ag, DNP-human serum albumin (HSA) (Sigma-Aldrich).

β-hexosaminidase released by FcεRI-triggered mast cell degranulation was measured by a colorimetric assay as previously described (11, 13). In brief, 2 × 10⁵ cells/sample were sensitized with IgE overnight and stimulated with DNP-HSA for 30 min. Samples from the supernatant and total cell lysates were analyzed. The amount of β-hexosaminidase release (in the supernatant) was determined by measuring the OD at 400 nm and represented as a percentage of total enzyme.

Supernatants from mast cells (2 × 10⁶ cells/sample) were collected 24 h after FcεRI stimulation and stored at −20°C until use. TNF-α, IL-1β, IL-3, IL-5, IL-6, IFN-γ, IL-8, MCP1, MCP1, MIP1α, MIP1β, eotaxin-1, and eotaxin-2, were evaluated by ELISA (R&D Systems) or using a multiplex immunoassay (Cytokine 20-Plex, BioSource) with the Bio-Plex System (Bio-Rad) following the manufacturer’s instructions.

Cytosolic calcium was measured as described previously (11, 13). In brief, IgE-sensitized cells were loaded with fura2-AM; labeled cells were placed in a prewarmed cuvette at 37°C in a spectrofluorimeter chamber. DNP-HSA was added to the cuvette and the release of calcium from internal stores was monitored by the changes in fluorescence.

SphK activity was measured by a radiometric assay as described previously (11, 13).

PGE₂ production was measured by the Biotrak PGE₂ system (Amersham Biosciences, GE Healthcare).

LTB₄ production was measured by the Biotrak leukotriene B₄ enzyme immunoassay system (Amersham Biosciences, GE Healthcare).

NFκB activity was analyzed using the Mercury TransFactor-“Profiling Kit-Inflammation” (BD Biosciences) following the manufacturer’s instructions and as previously described (18).

Forty micrograms of protein from total cell lysates/samples were resolved in 10% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 μm nitrocellulose membranes as previously stated (11, 13). The blots were probed using the polyclonal anti-SphK1 made in-house and used as previously described (11); against SphK2 (Exalpha Biologicals); against α-tubulin (Upstate Biotechnology) for loading control. Bands were visualized using anti-IgG HRP-conjugated secondary Abs, and the ECL Western Blotting Detection System (GE Healthcare).

Experiments were performed in two strains (BALB/c, and 129Sv × C57BL6 mixed background) 8- to 10-wk-old mice obtained from the Laboratory Animal Holding Unit, National University of Singapore. The study was conducted in accordance with the University of Singapore Guidelines for animal experimentation. Mice were lightly anesthetized and injected i.v. with the siRNA, following a protocol we recently developed (19); for the injections, the tail was immersed in warm water (40°C) for 10 s to dilate the tail veins. Synthetic siRNA (4 μg of siRNA in 0.2 ml of PBS) was injected into one of the lateral tail veins using a 30G needle. The siRNA injection was repeated after 24 and 48 h, to achieve a significant SphK gene-silencing effect. The siRNA-sequences were the same as for the in vitro experiments.

Mice were anesthetized and injected s.c. in the dorsal skin with mouse anti-DNP IgE (20 ng in 20 μl saline). Saline alone was used as negative control. Mice were injected i.v. 24 h later with 100 μg of DNP-HSA with 1% Evans blue dye. Vascular permeability was visualized 60 min later by the blue staining of the injection sites on the reverse side of the skin. Skin samples were also harvested and Evans blue extracted from the tissue, using 0.5 ml of formamide at 55°C for 48 h and quantified by OD at 610 nm.

Mice were injected i.v. in the tail vein with mouse anti-DNP IgE (200 μg in 200 μl saline). Saline alone was used as negative control. Twenty-four hours later, mice were challenged with 100 μg of DNP-HSA. Changes in body temperature associated with systemic anaphylaxis were monitored every 10 min, by measuring changes in rectal temperature, using a rectal probe coupled to a digital thermometer (Natsume Seisakusyo). At the end of the experiment (120 min after challenge), mice were sacrificed and plasma collected and stored at −20°C until used. IL-6, IL-3, and TNF-α protein levels in the plasma were evaluated using ELISA (R&D Systems) following the manufacturer’s instructions. Histamine levels in the plasma were evaluated using ELISA (IBL), following the manufacturer’s instructions. Portions of lungs were excised and fixed with 10% buffered formamide (Sigma-Aldrich), dehydrated, and embedded in paraffin for histological examination. Sections (5 μm) were stained with H&E and evaluated by light microscopy and documented by photographs.

Skin samples were fixed in 10% formalin and 4-μm paraffin sections were stained with Toluidine blue and examined with an Olympus microscope (model BX 51) using ×10, ×40, and ×100 uplan Apo lenses. Mast cell numbers in the skin were calculated by counting the cells in six different sections, derived from six mice, under light microscopy. The percentage of degranulated mast cells in the skin was calculated by observing the number of cells with >10% extrusion of granules as previously described (20, 21).

Statistical differences between control and treatment groups were calculated using unpaired Student’s t test.

Knockout mice were generated by Genoway. In brief, for SPHK1 knockout, a targeting vector for homologous recombination was generated that contained at the 5′ end a diphteria toxin A negative selection cassette to favor the homologous recombination event, a 5′ homologous region consisting of 2.8kb homologous SPHK1 sequences including exon 1 and 2, a Neo-cassette instead of exons 3–6, and a 3′ homologous region consisting of 5.9kb of the subsequent genomic sequence.

For SPHK2 knockout, a targeting vector was generated that contained at the 5′ end a diphteria toxin A negative selection cassette, a 5′ homologous region consisting of 6.1kb homologous SPHK2 sequences including exons 1–3, a Neo-cassette flanked by two LoxP sites instead of exons 4–7, and a 3′ homologous region consisting of 1.8kb targeted SPHK2 sequences including exon 8.

Gene targeted embryonic stem cells were isolated and used to produce chimeric male mice which passed the disrupted allele to their offsprings. Heterozygous matings produced viable mice homozygous for the mutation. Mice were backcrossed to C57BL/6 in an accelerated backcrossing procedure by Charles River Laboratories.

We first stimulated murine mast cells by FcεRI cross-linking and looked at the subcellular localization of the SphK isoforms, and found that FcεRI cross-linking triggers the translocation of SphK1 from the cytosol to the plasma-membrane periphery; however, the localization of SphK2 remained unaltered (Fig. 1,a). We then went on to specifically knockdown SphK1 or SphK2 by RNA-interference (Fig. 1,b), and measured SphK activity and S1P generation: FcεRI-triggered SphK and S1P was substantially reduced in cells where SphK1 had been knocked down, whereas silencing SphK2 had no effect on the FcεRI-mediated SphK activity (Fig. 1, c and d). After which, we investigated the role of each of the SphK-isoforms in FcεRI-mediated activation of intracellular signaling pathways essential for mast cell degranulation, such as FcεRI cell surface expression, calcium mobilization, and the release of granular contents. Using siRNA knockdown, we show that neither SphK1 nor SphK2 is required for FcεRI cell surface expression (Fig. 2,a). However, SphK1 was essential for the FcεRI-triggered initial rise in cytosolic calcium signals (Fig. 2,b) and also for mast cell degranulation (Fig. 2,c), whereas silencing SphK2 had no effect at all on any of the FcεRI-mediated responses (Fig. 2). FcεRI stimulated human mast cells to produce substantial amounts of PGD₂ and LTC₄ in a SphK1-dependent manner (Fig. 3, a and b), both of which are important lipid-derived mediators for mast cell-mediated responses. FcεRI triggered a substantial rise of cytokines, including IL-1β, IL-3, IL-5, IL-6, and TNF-α, and chemokines MIP-2, eotaxin-2, MCP-1, MIP-1α, and MIP1-β (Fig. 3, c and d). The production of several of the cytokines and chemokines was inhibited in cells pretreated with the SphK1-siRNA, whereas consistent with other results, the siRNA-SphK2 had no effect at all. These results were observed in mast cells derived from BALB/c and 129Sv × C57BL6 mice.

We investigated the effect of SphK-silencing on the activities of NFκB. FcεRI triggers the activation of NFκB; however, in cells pretreated with the siRNA specific for SphK1, but not that for SphK2, the FcεRI-stimulated NFκB activity was abrogated (Fig. 3e). In addition, the SphK1-siRNA prevented the generation of IL-1β, IL-3, IL-6, TNF-α, MIP-2, MCP-1, MIP-1α, and MIP-1β synthesis, whereas the SphK1-siRNA had no effect on FcεRI-triggered IL-5 or eotaxin-2 synthesis (Fig. 3, c and d). The siRNA for SphK2 had no effect at all. The results were similar in mast cells from BALB/c and 129Sv × C57BL6 mice.

We then silenced either SphK1 or SphK2, by siRNA in vivo transfection in adult mice as recently described (19), in two different backgrounds of mice, namely, in BALB/c and 129Sv × C57BL6 mice (Fig. 4,a). We then investigated the role of each SphK-isoform in an anaphylactic response using the model of IgE-mediated passive cutaneous anaphylaxis (PCA) (22), the gold standard model of mast cell-mediated hypersensitivity. One of the key inflammatory events during an allergic reaction is an increase in vascular permeability (20, 21, 22). Thus, we sensitized mice s.c. with anti-DNP IgE Ab and then challenged i.v. 24 h later with DNP-HSA in saline-containing Evans blue to measure vascular permeability. The amount of Evans blue in control and challenged mice was quantified and showed the expected increase in vascular permeability after antigenic challenge. The PCA-triggered vascular permeability was considerably inhibited in mice pretreated with the SphK1-siRNA, whereas the PCA-response in mice pretreated with the SphK2-siRNA showed a similar level of vascular permeability as the positive control mice. Further analysis on the Evans blue levels in the skin confirmed the visual observation and revealed a significant decrease of vascular permeability only in mice pretreated with the SphK1-siRNA; similar results were observed in both mouse strains (Fig. 4,b). To further test whether SphK1 was critical for mast cell degranulation in vivo, skin samples at the injection sites were removed and examined histologically. The level of mast cell degranulation was very much reduced in the SphK1-silenced mice, whereas the siRNA-SphK2-silenced mice displayed similar responses to that of the positive control group; similar results were observed in both mouse strains (Fig. 4 c).

We then investigated the role of silencing SphK1 or SphK2 in systemic anaphylactic shock. BALB/c and 129Sv × C57BL6 mice, which were injected i.v. with anti-DNP IgE Ab and challenged i.v. 16 h later, developed markedly reduced body temperature, reaching 30°C 30 min after antigenic challenge (Fig. 5,a). The rapid decrease in body temperature was considerably reduced in mice pretreated with the SphK-1 siRNA. However, in mice pretreated with the SphK2-siRNA the drop in temperature was identical with the positive control mice (Fig. 5,a). The effect of specifically silencing SphK1 in the induction of anaphylactic shock was also reflected in the plasma cytokine and chemokine concentrations (Fig. 5,b), and histamine levels (Fig. 5 c); top panels show data from BALB/c mice, whereas bottom panels show data from 129Sv × C57BL6 mice. Taken together, these data show that mice pretreated with the SphK1-siRNA were largely protected from IgE-mediated anaphylaxis.

As our data using the siRNA are in sharp contrast with a recently published work using SPHK1^−/− and SPHK2^−/− mice (15), we performed experiments in mast cells derived from SPHK1^−/− and SPHK2^−/− mice. Firstly, we show that mast cells derived from either KO mice do not express the relevant SphK-isoforms (Fig. 6,a), we then show that the calcium response and degranulation, triggered by FcεRI-cross-linking, is similar in wild-type and mast cells derived from either knockout mice (Fig. 6, b and c). We then investigated the IgE-mediated anaphylaxis in the knockout mice, we show that either mice is negative for the corresponding KO-SphK (Fig. 6,d), and found that following the induction of systemic anaphylaxis, similar temperature changes and serum histamine levels, were observed for both knockout mice as well as for the wild-type mice (Fig. 6, e and f), indicating that no protection was observed for either knockout mice.

Taken together, these data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and that silencing SphK1 in adult mice using siRNA, clearly establishes a role for SphK1, but not SphK2, in the IgE-mediated mast cell degranulation and anaphylaxis.

Recently, it has been suggested that FcεRI, activates both SphK1 and SphK2 to drive different responses in mouse and human mast cells (15, 16). However, this is in contradiction with previous reports showing that SphK1 is activated by FcεRI, and mediates proinflammatory responses, in rat, mouse, and human mast cells (11, 13). In this study, we show that only SphK1 is activated by FcεRI in mast cells derived from two different strains of mice, BALB/c, and 129Sv × C57BL6 mice, and provide solid evidence for the specific role of SphK1 in FcεRI-mediated calcium mobilization and degranulation in vitro. During an anaphylaxis FcεRI-mediated mast cell activation results in degranulation, and sustained de novo production of a number of cytokines and eicosanoids, which contribute to the pathology underlying allergic disease (20, 21, 22, 23, 24). As SphK1 is a key enzyme in degranulation and elevates the cytoplasmic Ca²⁺ levels necessary for cPLA₂ and for NFκB activation, we investigated the ability of SphKs to mediate the production of eicosanoids and cytokines. FcεRI triggered a substantial rise of the eicosanoids PGE₂ and LTC₄, as well as the cytokines IL-1β, IL-3, IL-5, IL-6, and TNF-α, and chemokines MIP-2, eotaxin-2, MCP-1, MIP-1α, and MIP-1β. The production of PGE₂, LTC₄, and of several of the cytokines and chemokines was inhibited in cells pretreated with the SphK1-siRNA, whereas consistent with other results, the siRNA-SphK2 had no effect at all.

Taken together, these results therefore demonstrate that SphK1, but not SphK2, is able to mediate a wide range of mast cell-mediated responses. It has been suggested that while the production of proinflammatory cytokines in mast cells is NFκB (p50 and p65) dependent (25, 26), the induction of Th2-like cytokines is NFκB independent (27)_. We therefore investigated the effect of SphK silencing on the activities of NFκB. FcεRI triggers the activation of NFκB; however, in cells pretreated with the siRNA specific for SphK1, but not that for SphK2, the FcεRI-stimulated NFκB activity was abrogated. This is in agreement with an earlier observation that Ca²⁺ signal amplitude and frequency play a role in NFκB activation (28). Consistent with this, the SphK1-siRNA prevented the generation of IL-1β, IL-3, IL-6, TNF-α, MIP-2, MCP-1, MIP-1α, and MIP-1β synthesis, which are dependent on NFκB activity (25), whereas the SphK1-siRNA had no effect on FcεRI-triggered IL-5 or eotaxin-2 synthesis, which is independent of the NFκB pathway (27).

Taken together, our in vitro data is in agreement with previous studies in rat, mouse, and human mast cells, which showed that SphK1 is critical for mast cell-mediated responses (11, 13). However, the relevance of these in vitro studies to the physiological role of SphK1 is unclear as a recent study, using murine mast cells from SPHK1^−/− and SPHK2^−/− mice, showed that SphK2 is the important isotype for murine mast cell-mediated responses (15).

Genetic robustness enables any organism to tolerate changes in its environment or genetic composition (29). The mechanisms may be explained by genetic buffering, where alternative mechanisms are present for a particular biological process in an organism, or by functional complementation, where genes are at some point redundant in their particular biological task due to the presence of two or more isoforms (30, 31). Two genes are said to be redundant if they can partially, or fully, compensate with each other functions and it may be considered that the genetic redundancy gives an organisms an advantage, as having two genes performing similar biological tasks, being able to compensate for each other should one of the genes be mutated (32). In our case, the SphK isoforms execute a similar task, but under normal circumstances their regulation may be different and their activation may be differentially modulated in different signal-transduction pathways. However, as Nowak et al. (33) mentioned on the evolution of genetic redundancy, we may conclude that the presence of SphK isoforms would certainly increase the robustness of the knock out mice to function normally in more than one state or situation (34), and this could well be the reason why our data are in sharp contrast with previous reports that demonstrate a clear dependence on SphK1 expression for mast cell-mediated responses. Thus, suggesting that possible adaptation and/or the existence of compensatory mechanisms during embryonic development of the knockout mice, because complete deficiency of SphK activity in double knockout is embryonic lethal, but mice lacking either isoform appear normal (34, 35).

To overcome this genetic robustness in the SPHK1 and SPHK2 KO mice, we have adopted RNA-interference technology to assess the individual role of each SphK isoform in wild-type mice. We selectively and specifically silenced the SPHK1 or SPHK2 expression using siRNA (19), and investigated the specific roles of SphK1 or SphK2 in passive cutaneous and systemic anaphylactic shock in both BALB/c and 129Sv × C57BL6 mice. The rapid cutaneous and systemic inflammatory responses were considerably inhibited in SphK1-silenced mice. However, in SPHK2-silenced mice all the inflammatory parameters were identical with the positive control mice. Interestingly, we show in this study that in the SPHK1^−/− and SPHK2^−/− mast cells and mice the FcεRI-mediated degranulation and the IgE-mediated anaphylaxis are unchanged, indicating marked differences between the siRNA-gene silencing and the knockout mice.

In conclusion, the results presented in this study demonstrated that SphK1 is a key mediator of mast cell degranulation, leading to anaphylaxis. Moreover, our results show another important finding, that is, the observation that siRNA methodology can generate results different to those of gene-knockout methods, a phenomenon which has not been well-documented. The expanding view on the roles of mast cells in innate and adaptive immune responses, suggests a potential role for mast cells in various diseases including autoimmune diseases (36). Thus, suppression of mast cell function by SphK1 blockage offers a new potential therapeutic approach for diseases, such as allergic anaphylaxis and other diseases where mast cell activation may play a pathological role.

We thank A-K Fraser-Andrews for proofreading the manuscript.

The authors have no financial conflict of interest.