The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering

Abstract

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.

Figure 1

A strong SK activity mediates the rapid degradation of S in CPII mast cells. (A) Determination of SK activity of either nonstimulated (nst) or 2 min–stimulated (IgE/Ag) CPII cells. −, reactions without exogenous S. The position of S1P on the thin layer plate is indicated by an arrow (right). (B) Ser-containing lipids from nonstimulated (nst) or stimulated (IgE/Ag) CPII cells. The position of S, which served as a standard (Std), is indicated by an arrow (right). (C) S phosphorylation and degradation (100 nM or 10 μM as indicated) after incubating nonstimulated and stimulated cells (indicated at the bottom) for various time points (top). −, nonstimulated cells. Stimulated cells were preincubated with S for 1 h before stimulation. S served as a standard (Std); d, degradation product. The positions of S, S1P, and the degradation product are indicated by arrows (right).

Figure 1

A strong SK activity mediates the rapid degradation of S in CPII mast cells. (A) Determination of SK activity of either nonstimulated (nst) or 2 min–stimulated (IgE/Ag) CPII cells. −, reactions without exogenous S. The position of S1P on the thin layer plate is indicated by an arrow (right). (B) Ser-containing lipids from nonstimulated (nst) or stimulated (IgE/Ag) CPII cells. The position of S, which served as a standard (Std), is indicated by an arrow (right). (C) S phosphorylation and degradation (100 nM or 10 μM as indicated) after incubating nonstimulated and stimulated cells (indicated at the bottom) for various time points (top). −, nonstimulated cells. Stimulated cells were preincubated with S for 1 h before stimulation. S served as a standard (Std); d, degradation product. The positions of S, S1P, and the degradation product are indicated by arrows (right).

Figure 3

S inhibits the MAPK pathways. Cells were either left unstimulated (nst) or were pretreated with solvent before activation with IgE/Ag (IgE/Ag) or pretreated with S (IgE/Ag + S) for 1 h before activation with IgE/Ag. (A) Raf kinase activity was determined after immunoprecipitations. MBP was used as substrate (indicated by an arrow). Reactions were separated by SDS-PAGE. Western blots of the immunoprecipitate for normalization are shown below (raf IP, indicated by an arrow). (B) MEK 1 activity was determined after immunoprecipitations. MBP was used as a substrate (indicated by an arrow). Reactions were separated by SDS-PAGE. Western blots of the immunoprecipitate for normalization are shown below (MEK 1 IP, indicated by an arrow) (C) Western blots to investigate erk 1,2 activation/phosphorylation. Cells were activated for 10 min. erk 1,2 and phospho-erk 1,2 are indicated by arrows (right). (D) Western blots for jnk 1,2 activation/phosphorylation. Cells were stimulated for 10 min. jnk 1,2 and phospho-jnk 1 are indicated by arrows (right).

Figure 2

High levels of intracellular S strongly inhibit leukotriene synthesis and cytokine transcription. Cells were either left unstimulated (nst) or were pretreated with solvent before activation with IgE/Ag (IgE/Ag) or pretreated with S (IgE/Ag + S) for 1 h before activation with IgE/Ag. (A) Hexosaminidase release as indicated on the y-axis. Stimulation conditions are given on the x-axis. Values represent mean of quadruplicate experiments ± SD. (B) Leukotriene (LT) release as indicated on the y-axis (in pg/ml LT). Stimulation conditions are given on the x-axis. Values represent mean of quadruplicate experiments ± SD. (C) The transcriptional activation of IL-5 and TNF-α was determined in a reporter gene assay after transient transfections. pGL2 represents the parental vector. Corrected luciferase values are given on the y-axis; stimulation conditions and input DNA are indicated on the x-axis. Data represent mean ± SD of triplicate determinations.

Figure 2

High levels of intracellular S strongly inhibit leukotriene synthesis and cytokine transcription. Cells were either left unstimulated (nst) or were pretreated with solvent before activation with IgE/Ag (IgE/Ag) or pretreated with S (IgE/Ag + S) for 1 h before activation with IgE/Ag. (A) Hexosaminidase release as indicated on the y-axis. Stimulation conditions are given on the x-axis. Values represent mean of quadruplicate experiments ± SD. (B) Leukotriene (LT) release as indicated on the y-axis (in pg/ml LT). Stimulation conditions are given on the x-axis. Values represent mean of quadruplicate experiments ± SD. (C) The transcriptional activation of IL-5 and TNF-α was determined in a reporter gene assay after transient transfections. pGL2 represents the parental vector. Corrected luciferase values are given on the y-axis; stimulation conditions and input DNA are indicated on the x-axis. Data represent mean ± SD of triplicate determinations.

Figure 4

AP1 transcription is prevented by S. (A) Western blots for c-jun activation/phosphorylation were performed with lysates of either nonstimulated (nst), 1 h–stimulated (IgE/Ag), or 1 h S-pretreated cells before activation (IgE/Ag + S). c-jun and phospho-c-jun are indicated by arrows (right). (B) The transcriptional activation of an AP1-dependent reporter gene (3 x TRE) was measured in transient transfections. Corrected luciferase values are indicated on the y-axis, stimuli and input DNA on the x-axis. pGL2 represents the parental vector. Cells were activated for 3 h. Data represent mean ± SD of triplicate determinations.

Figure 6

Effects of S and S1P on BMMCs. (A) Ser-containing lipids from nonstimulated (nst) or 1 h–stimulated (IgE/Ag) BMMCs. The position of S, which served as a standard (Std), is indicated by an arrow (right). (B) Determination of SK activity of either nonstimulated (nst) or 2 min–stimulated (IgE/Ag) BMMCs. −, reactions without exogenous S. The position of S1P on the thin layer chromatography plate is indicated by an arrow (right). (C) S phosphorylation and degradation (100 nM or 10 μM as indicated) after incubating nonstimulated and stimulated cells (indicated at the bottom) for various time points (top); −, nonstimulated cells. Stimulated cells were preincubated with S for 1 h before stimulation. S served as a standard (Std); d, degradation product. The positions of S, S1P, and the degradation product are indicated by arrows (right). (D) The secretion of TNF-α by BMMCs was determined by a corresponding ELISA. pg/ml TNF-α are indicated on the y-axis, stimulation conditions on the x-axis. Cells were either left unstimulated (nst) or were pretreated with solvent, 10 μM S (S), or 10 μM S plus 10 μM S1P (S + S1P) before activation with IgE/Ag for 6 h. Data represent mean ± SD of quadruplicate determinations. (E) Western blots to investigate erk 1,2 activation/phosphorylation. Cells were activated for 10 min. erk 1,2 and p-erk 1,2 are indicated by arrows (right).

Figure 5

S1P reverts S inhibition of TNF-α. (A) The transcriptional activation of a TNF-α reporter gene construct was determined in transient transfections. Corrected luciferase values are indicated on the y-axis, stimulation conditions on the x-axis. Cells were either left unstimulated (nst) or were pretreated with solvent, 10 μM S (S), or 10 μM S plus 10 μM S1P (S + S1P) before activation with IgE/Ag. Data represent mean ± SD of triplicate determinations. (B) Determination of S in stimulated cells (time points are given above the panels; −, nonstimulated cells) incubated either with S alone or S plus S1P as indicated at the top by thin layer chromatography. The positions of S, S1P, and degradation product (d) are indicated with arrows (right). S served as standard (Std).

Figure 7

S1P is an activator of CPII mast cells. (A) Hexosaminidase release assay with either nonstimulated (nst) or 1 h–stimulated cells. Hexosaminidase values are indicated on the y-axis, stimuli on the x-axis. Values represent mean of quadruplicate experiments ± SD. (B) Leukotriene (LT) release of nonstimulated (nst) or 4 h–stimulated cells. pg/ml LT are indicated on the y-axis, stimuli on the x-axis. Data represent mean of quadruplicate determinations ± SD. (C) TNF-α reporter gene assay. Cells were either left unstimulated (nst) or were pretreated with solvent before stimulation with IgE/Ag, or stimulated with 10 μM S1P, 100 ng/ml ionomycin, and 20 ng/ml PMA. Data represent mean ± SD of triplicate determinations. Luciferase values are indicated on the y-axis, stimuli on the x-axis.

Figure 7

S1P is an activator of CPII mast cells. (A) Hexosaminidase release assay with either nonstimulated (nst) or 1 h–stimulated cells. Hexosaminidase values are indicated on the y-axis, stimuli on the x-axis. Values represent mean of quadruplicate experiments ± SD. (B) Leukotriene (LT) release of nonstimulated (nst) or 4 h–stimulated cells. pg/ml LT are indicated on the y-axis, stimuli on the x-axis. Data represent mean of quadruplicate determinations ± SD. (C) TNF-α reporter gene assay. Cells were either left unstimulated (nst) or were pretreated with solvent before stimulation with IgE/Ag, or stimulated with 10 μM S1P, 100 ng/ml ionomycin, and 20 ng/ml PMA. Data represent mean ± SD of triplicate determinations. Luciferase values are indicated on the y-axis, stimuli on the x-axis.

Figure 8

S1P induces the MAPK pathway and AP1 transcription factors. (A) Western blot analysis with lysates of nonstimulated (nst) or 10 μM S1P–stimulated cells for the time points indicated. MEK 1,2, phospho-MEK 1,2, erk 1,2, p-erk 1,2, jnk 1,2, p-jnk 1, c-jun, and p-c-jun are indicated by arrows (right). (B) Supershift analysis on an AP1 consensus site as radiolabeled probe with nuclear extracts of either nonstimulated (nst) or 1 h S1P–stimulated cells as indicated at the top. f, free probe. Antibodies against AP1 transcription factors are given at the top. −, activated complex without antibody.

Figure 9

Activated mast cells secrete S1P. (A) Lipids detected in the supernatants of nonstimulated (nst) or stimulated (IgE/Ag) CPII cells that were fed with ¹⁴C-Ser for 24 h. After extraction, lipids were separated by thin layer chromatography. S and S1P served as standard (Std); the positions of S, S1P, and the degradation product (d) are indicated by arrows (right). (B) Sphingolipids detected in supernatants of nonstimulated or stimulated (IgE/Ag) CPII cells that were fed with ³H-S for 1 h before activation. Time points of activation are indicated at the top; control is the cellular content of sphingolipids. S and S1P served as standard. The positions of S, S1P, and the degradation product (d) are indicated by arrows (right). (C) Lipids detected in the supernatants of nonstimulated (nst) or stimulated (IgE/Ag) BMMCs that were fed with ¹⁴C-Ser for 24 h. After extraction, lipids were separated by thin layer chromatography. S and S1P served as standard (Std); the positions of S, S1P, and the degradation product (d) are indicated by arrows (right). (D) Hexosaminidase release assay with either nonstimulated (nst) or 1 h–stimulated BMMCs. Hexosaminidase values are indicated on the y-axis, stimuli on the x-axis. Values represent mean of quadruplicate experiments ± SD.