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

Abstract

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcεRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcεRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcεRI stimulation including: Ca2+ signals, NFκB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1−/− and SPHK2−/− mice, which showed that SphK2 was required for FcεRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1−/− and SPHK2−/− mice and show that the calcium response and degranulation, triggered by FcεRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

The SNARE Machinery in Mast Cell Secretion

“…As already mentioned above, we and others found in BMMC that absence of VAMP8 did not affect secretion of several chemokines or cytokines tested contrasting with some recent data showing an implication of VAMP8 in anaphylatoxin-induced TNF release in macrophages (Tiwari et al, 2008; Pushparaj et al, 2009). For the latter its implication in cytokine release was specific for TNF as IL-1β, IL-6, and CCL3 were not affected.…”

“…For the latter its implication in cytokine release was specific for TNF as IL-1β, IL-6, and CCL3 were not affected. In wild-type macrophages, TNF was found to colocalize with VAMP8-positive vesicles, and in VAMP8-deficient macrophages, TNF release was inhibited (Tiwari et al, 2008; Pushparaj et al, 2009). Furthermore, VAMP8 has been shown to regulate the release of TNF and β-hexosaminidase in macrophages triggered by fMLP (Alvarez de Toledo and Fernandez, 1990).…”

The Src Family Kinase Fgr Is Critical for Activation of Mast Cells and IgE-Mediated Anaphylaxis in Mice

“…An incidental finding was that the PCA reaction was also associated with an increase in mast cell number in the targeted ear in the mice by stimulating with antigen (Fig. 8E and see reference 36). Although it is unlikely that this could be attributed to formation of additional mast cells from progenitor cells because of the relatively short period after injection of antigen (i.e.…”

Regulation of Mammalian Physiology, Development, and Disease by the Sphingosine 1-Phosphate and Lysophosphatidic Acid Receptors

“…For instance, mast cells produce and secrete S1P, which is thought to bind S1PR to modulate FcεR-mediated degranulation and inflammatory mediator synthesis, and in vivo studies using Sphk1 -/- or Sphk2 -/- mice yield conflicting results 46,428,429 . Similarly, in studies utilizing preadipocytes, LPA binding to LPA 1 repressed PPARγ in vitro, and mice with adipocyte-specific deletion of ATX demonstrated increased adipocity with a concomitant increase in PPARγ expression, implying that the LPA that affects PPARγ is produced extracellularly and signals via LPAR 430,431 .…”