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

Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T - J. Exp. Med. (1999)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Novartis Research Institute, Vienna, Austria. eva.prieschl@pharma.novartis.com

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.

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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).
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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).

Mentions: PMA-dependent PKCs, the prime targets for inhibition by lysosphingolipids, are known to be dispensable for the induction of TNF-α in CPII cells after Fc∈RI triggering 16. The production of this cytokine and leukotriene synthesis strongly depend on the activation of the MAPK pathway, suggesting that this signaling cascade is a previously unrecognized target for S-mediated modulation 18. 1-h treatment with 10 μM S before IgE/Ag activation prevented the 50–100-fold enhancement of the raf and MEK kinase activity, measured in in vitro kinase reactions of immunoprecipitates using myelin basic protein (MBP) as a readout (Fig. 3A and Fig. B). Subsequently, the two IgE/Ag-responsive MAPKs in CPII cells, erk 1,2 and jnk 1, are hypophosphorylated, as measured in a Western blot analysis (pThr 202/pTyr 204 and pThr 183/pTyr 185; Fig. 3C and Fig. D). This shows that the inhibitory potential of S affects all MAPK pathways that are induced in mast cells after IgE/Ag stimulation 16.


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

Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T - J. Exp. Med. (1999)

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).
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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).
Mentions: PMA-dependent PKCs, the prime targets for inhibition by lysosphingolipids, are known to be dispensable for the induction of TNF-α in CPII cells after Fc∈RI triggering 16. The production of this cytokine and leukotriene synthesis strongly depend on the activation of the MAPK pathway, suggesting that this signaling cascade is a previously unrecognized target for S-mediated modulation 18. 1-h treatment with 10 μM S before IgE/Ag activation prevented the 50–100-fold enhancement of the raf and MEK kinase activity, measured in in vitro kinase reactions of immunoprecipitates using myelin basic protein (MBP) as a readout (Fig. 3A and Fig. B). Subsequently, the two IgE/Ag-responsive MAPKs in CPII cells, erk 1,2 and jnk 1, are hypophosphorylated, as measured in a Western blot analysis (pThr 202/pTyr 204 and pThr 183/pTyr 185; Fig. 3C and Fig. D). This shows that the inhibitory potential of S affects all MAPK pathways that are induced in mast cells after IgE/Ag stimulation 16.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Novartis Research Institute, Vienna, Austria. eva.prieschl@pharma.novartis.com

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.

Show MeSH