Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

Mast cells are immunoregulatory cells that participate in inflammatory processes. Cross-linking mast cell specific GD1b derived gangliosides by mAbAA4 results in partial activation of mast cells without the release of preformed mediators. The present study examines the release of newly formed and newly synthesized mediators following ganglioside cross-linking. Cross-linking the gangliosides with mAbAA4 released the newly formed lipid mediators, prostaglandins D2 and E2, without release of leukotrienes B4 and C4. The effect of cross-linking these gangliosides on the activation of enzymes in the arachidonate cascade was then investigated. Ganglioside cross-linking resulted in phosphorylation of cytosolic phospholipase A2 and increased expression of cyclooxygenase-2. Translocation of 5-lipoxygenase from the cytosol to the nucleus was not induced by ganglioside cross-linking. Cross-linking of GD1b derived gangliosides also resulted in the release of the newly synthesized mediators, interleukin-4, interleukin-6, and TNF-α. The effect of cross-linking the gangliosides on the MAP kinase pathway was then investigated. Cross-linking the gangliosides induced the phosphorylation of ERK1/2, JNK1/2, and p38 as well as activating both NFκB and NFAT in a Syk-dependent manner. Therefore, cross-linking the mast cell specific GD1b derived gangliosides results in the activation of signaling pathways that culminate with the release of newly formed and newly synthesized mediators.


Introduction
Gangliosides are sialic acid containing glycosphingolipids that are present in the outer leaflet of the plasma membrane as well as in the membranes of some organelles [1,2]. Gangliosides play a role in diverse physiological processes including growth, differentiation, cell-cell interactions, and cell signaling. They are also involved in many pathological processes, acting as receptors for viruses and toxins, and are implicated in tumor progression, atherosclerosis, and neurodegenerative disorders [3].
Gangliosides are present on the surface of mast cells and are critical for mast cell function [1]. Mast cells are multifunctional immune cells that participate in various biological events, such as inflammation and allergy. Mast cell functions are directly related to their activation and subsequent release of biologically active mediators [4,5]. Mast cell activation via the high affinity IgE receptor (Fc RI) is the best characterized form of activation. It occurs when multivalent antigens cross-link antigen-specific immunoglobulin E (IgE) bound to Fc RI on the mast cell surface. Crosslinking Fc RI initiates a signal transduction cascade that is dependent on the tyrosine kinase Syk [6]. This activation results in the release of three classes of mediators: preformed mediators such as histamine, proteases, cytokines, and enzymes; newly formed lipid mediators which are 2 Mediators of Inflammation comprised of prostaglandins (PG), leukotrienes (LT), and platelet activating factor; and newly synthesized mediators which include cytokines and chemokines [4,7,8].
Mast cell specific gangliosides derived from GD1b are present on the surface of rodent mast cells [9,10]. Crosslinking the GD1b derived gangliosides with a ganglioside specific monoclonal antibody (mAbAA4) or its F(ab ) 2 fragment results in partial activation of RBL-2H3 mast cells without degranulation or release of preformed mediators [11,12]. Although cross-linking GD1b derived gangliosides activates mast cells, whether or not cross-linking these gangliosides stimulates release of newly formed and newly synthesized mediators has not been investigated. Therefore, it was of interest to determine if newly formed lipid mediators and newly synthesized mediators were released following ganglioside cross-linking and whether or not this release was Sykdependent.

Mast Cell Activation and Mediator
Release. In order to cross-link the GD1b derived gangliosides, RBL-2H3 cells and C4A2 cells were incubated with mAbAA4 at various concentrations (1, 2.5, 5, or 10 g/mL) depending on the experiment. For stimulation via Fc RI, cells were sensitized overnight (ON) with mouse IgE anti-TNP ascites fluid (1 : 5,000 dilution) and then stimulated with 50 ng/mL of DNP 48 -HSA (Sigma-Aldrich) for 30 min or for 1 h and then rinsed and cultured for an additional 3 h, for the release of newly formed lipid mediators (PGD 2 , PGE 2 , LTB 4 , and LTC 4 ). In order to examine the release of newly synthesized mediators (IL-4, IL-6, and TNF-), cells were stimulated for 1 h, rinsed, and cultured for an additional 11 h. For Fc RI independent stimulation, cells were incubated with 0.1 g/mL of calcium ionophore A23187 (Sigma-Aldrich). PGD 2 , PGE 2 , LTB 4 , and LTC 4 in culture supernatants were analyzed using EIA kits (Cayman Chemical, Ann Arbor, MI). IL-4, IL-6, and TNF-in the culture supernatants were measured using ELISA kits (BD Biosciences) according to the manufacturer's instructions. Nonstimulated cells were used as controls.

NF B and NFAT
Activation. NF B2 cells, VB9 cells, IC2 cells, and IH10 cells were incubated with mAbAA4, stimulated via Fc RI, or with calcium ionophore for 1 h (as described in Section 2.3), rinsed, and cultured for an additional 5 h (NF B activation) or 15 h (NFAT activation). Cells were analyzed by flow cytometry and the percent of GFP positive cells was determined using a Guava Easy Cyte Mini System and Cytosoft Blue software (Guava Technologies Inc., Hayward, CA).

Immunoblotting.
Total cells lysates were obtained as previously described [17]. For some experiments, nuclear and cytosolic extracts were obtained as previously described [18]. The proteins were separated electrophoretically on 8% or 12% polyacrylamide gels and transferred to Hybond membranes (GE Healthcare Life Sciences, Marlborough, MA). After transfer, the membranes were blocked for 1 h at RT in TTBS (0.05 M Tris-HCl, 0.15 M NaCl, pH 7.5, and 0.05% Tween 20) containing 4% BSA (Sigma-Aldrich). After blocking, the membranes were incubated ON at 4 ∘ C with the primary antibodies diluted in TTBS. The membranes were then washed, incubated for 30 min with secondary antibody, and developed using enhanced chemiluminescence (ECL Kit; GE Healthcare). The images were obtained with ImageQuant LAS 4000 (GE Healthcare). Mean optical density of the target protein was determined using ImageJ software (NIH).

Statistical Analyses.
Results were analyzed using Graph-Pad Prism (GraphPad Software, Inc., San Diego, CA). The results were expressed as mean ± SD and differences between experimental samples were assessed by one-way analysis of variance (ANOVA) with Bonferroni's post hoc test; < 0.05 was considered statistical significant.

Cross-Linking GD1b Derived Gangliosides with mAbAA4
Induced the Release of Newly Formed Lipid Mediators PGD 2 and PGE 2 . RBL-2H3 cells and C4A2 Syk-negative cells

Cross-Linking GD1b Derived Gangliosides with mAbAA4
Resulted in Phosphorylation of Cytosolic Phospholipase A 2 (cPLA 2 ) and Cyclooxygenase-2 (COX-2) Expression. Crosslinking GD1b derived gangliosides resulted in the release of PGs but not LTs. Therefore, cPLA 2 phosphorylation and induction of COX-2 expression, which are required for PG generation, were investigated. An increase in cPLA 2 phosphorylation was observed after incubation of RBL-2H3 cells with mAbAA4 for 5 min and the levels of cPLA 2 phosphorylation were higher than those observed in cells stimulated via Fc RI (Figures 2(a) and 2(b)). COX-2 expression was also induced in cells incubated with mAbAA4 for 1 h and rinsed and cultured for an additional 3 h (Figures  2(c) and 2(d)). In contrast, translocation of 5-LO from the cytosol to the nucleus, a requirement for LT generation, was not induced by ganglioside cross-linking ( Supplementary  Figures 1(C)-1(F)). Therefore, the cross-linking of GD1b derived gangliosides specifically induces the activation of the arachidonic pathway responsible for PG generation in mast cells.

Cross-Linking GD1b Derived Gangliosides with mAbAA4
Induced the Release of Newly Synthesized Cytokines. Mast cell activation via Fc RI leads to transcription factor activation resulting in the production and release of cytokines [19]. Therefore, it was of interest to investigate whether cytokines are released after cross-linking GD1b derived gangliosides by mAbAA4. RBL-2H3 cells and Syk-negative C4A2 cells were incubated with mAbAA4 for 1 h and rinsed and cultured for an additional 11 h. Ganglioside cross-linking resulted in a Syk-dependent release of the newly synthesized mediators, interleukin-4 (IL-4) (Figure 3(a)), interleukin-6 (IL-6) (Figure 3(b)), and tumor necrosis factor-(TNF-) (Figure 3(c)). Interestingly, the amount of cytokines released after ganglioside cross-linking, with the exception of IL-6, was lower than that observed after stimulation via Fc RI.

Cross-Linking GD1b Derived Gangliosides with mAbAA4
Induced MAP Kinase Phosphorylation. MAP kinases are involved in signaling pathways that lead to production of newly synthesized mediators [20]. Since incubation of mast cells with mAbAA4 resulted in the release of IL-4, IL-6, and TNF-, it was of interest to investigate the degree of MAP kinase phosphorylation induced by cross-linking the GD1b derived gangliosides. When RBL-2H3 cells were incubated with mAbAA4 for 10 min, MAP kinases ERK1/2, JNK1/2, and p38 were phosphorylated (Figure 4). The degree of MAP kinase phosphorylation in mast cells incubated with mAbAA4 was less than that observed in cells stimulated via Fc RI, which agrees with the amount of cytokine released.

Cross-Linking GD1b Derived Gangliosides with mAbAA4
Induced the Activation of Transcription Factors. Transcription factor activation is the ultimate requirement for the production of newly synthesized mediators [21]. Therefore,  RBL-2H3 derived GFP reporter cell lines were used to assess NF B and NFAT activation. Cross-linking GD1b derived gangliosides by mAbAA4 induced activation of both NF B ( Figure 5(a)) and NFAT ( Figure 5(b)) in a Syk-dependent manner. However, transcription factor activation by ganglioside cross-linking was less prominent than that observed by stimulation via F RI.

Discussion
The present study demonstrates that cross-linking the mast cell specific GD1b derived gangliosides induces the release of newly formed and newly synthesized mediators. Furthermore, this release is Syk-dependent. However, previous investigations have demonstrated that mast cell activation by cross-linking these gangliosides does not induce the release of preformed mediators [11,12]. Moreover, other studies have shown that cross-linking gangliosides can also activate a variety of cell types [22][23][24][25][26]. Antibodies to gangliosides have been shown to activate PKC and increase proliferation in lymphocytes [27,28], stimulate calcium influx in oligodendrocytes [29], and induce leukocyte degranulation [30]. The molecular mechanisms by which cross-linking gangliosides can activate cells are poorly understood. Eicosanoids (prostaglandins, thromboxane, leukotrienes, and lipoxins) are the most important lipid mediators generated by mast cells [31]. The results of the present study show that cross-linking GD1b derived gangliosides induces release of prostaglandins, but not leukotrienes. Incubation with mAbAA4 stimulated cPLA 2 phosphorylation and incubation with mAbAA4 for extended periods of time increased COX-2 expression. The first step in eicosanoid generation is Ca 2+ -dependent phosphorylation of cPLA 2 through the MAP kinase pathway. Phosphorylated cPLA 2 translocates to cellular membranes, principally to the nuclear envelope, where arachidonic acid (AA) is released from membrane phospholipids by the action of cPLA 2 . AA is then metabolized either by COX-2 or CYP2E1 to produce PGs such as PGE 2 or by 5-LO to produce LTs in concert with 5-lipoxygenaseactivating protein (FLAP) on the nuclear envelope [32,33]. Previous studies have shown that the immediate phase of PG generation (5-30 min) requires the action of constitutively expressed COX-1 and phosphorylation of cPLA 2 , while the delayed phase of PG generation (4-6 h) depends on the induced expression of COX-2 [34]. In addition, cross-linking GD1b derived gangliosides did not induce 5-LO translocation from the cytosol to the nuclear membrane. These results agree with the findings that ganglioside cross-linking induces release of prostaglandins, but not leukotrienes, and indicate that ganglioside cross-linking selectively stimulates the eicosanoid biosynthetic pathway to induce PG generation.
Cross-linking GD1b derived gangliosides induces the release of the newly synthesized mediators IL-4, IL-6, and TNF-. In mast cells, newly synthesized mediator expression depends on the activation of signaling pathways that ultimately leads to transcription factor activation [21]. These events culminate with cytokine production and release and can occur even in the absence of mast cell degranulation [16]. A variety of studies investigating Fc RI independent mast cell activation also revealed that release of proinflammatory 8 Mediators of Inflammation mediators can occur in the absence of degranulation [35][36][37]. The production of newly synthesized mast cell mediators following Fc RI activation relies on MAP kinase signaling pathways as well as on the activation of the transcription factors NF B and NFAT.
The MAP kinase signaling pathway participates in activation, differentiation, proliferation, and migration of mast cells. Cross-linking GD1b derived gangliosides results in ERK1/2, JNK1/2, and p38 MAP kinase phosphorylation. ERK1/2 is an essential signal in the production of the newly synthesized mediators IL-5, IL-3, IL-13, and TNF-in mast cells [38]. JNK1/2 is responsible, at least partially, for the expression and production of several cytokines, including IL-6 and TNF-in mast cells [39]. Additionally, activation of p38 MAP kinase was shown to stimulate IL-4 production in bone marrow derived mast cells [40]. When mast cells are stimulated via Fc RI, the transcription factors NF B and NFAT are translocated to the nucleus and initiate the transcription of genes for proinflammatory and regulatory cytokines. This results in the expression and release of cytokines [19,41,42]. Cross-linking GD1b derived gangliosides by mAbAA4 activates the transcription factors NF B and NFAT. However, the degree of activation by ganglioside cross-linking was less than that observed by stimulation via Fc RI. This reduction in activation is expected since the degree of MAP kinase phosphorylation after ganglioside cross-linking was less than that observed in mast cells stimulated via Fc RI. Similar results have been reported for phosphorylation of Lyn, Syk, PLC 1, and the -and -subunits of Fc RI [12].
The lower phosphorylation of MAP kinase resulted in a reduction in NF B and NFAT activation leading to a decrease in IL-4 and TNF-release. In Fc RI stimulated mast cells, activation of NF B depends on PKC activation [43]. On the other hand, NFAT is activated by calcineurin induced dephosphorylation, a Ca 2+ -calmodulin dependent serine/threonine phosphatase that is activated by an increase in intracellular calcium [44,45]. mAbAA4 binding to RBL-2H3 mast cells results in a modest increase in intracellular calcium as well as in a partial redistribution of PKC [11], which could explain the reduced activation of NF B and NFAT seen in the present study. Additionally, cross-linking GD1b derived gangliosides in Syk-negative cells did not stimulate the release of either newly formed or newly synthesized mediators. This is in agreement with previous studies that have shown that the inhibition or the lack of Syk results in the failure of mast cells to produce and release any mediators [46,47]. Syk-negative mast cells are also unable to activate NF B and NFAT in response to Fc RI activation [6,16].
The exact mechanism by which cross-linking the GD1b derived gangliosides causes the various effects observed both previously and in this study is still unknown. Several intracellular signals induced by mAbAA4 binding are very similar to those induced by Fc RI activation. Binding of mAbAA4 to mast cells is known to stimulate protein tyrosine phosphorylation, including phosphorylation of Lyn, Syk, PLC 1, and the -and -subunits of Fc RI. However, the rate of phosphorylation of Lyn, Syk, and PLC 1 was slower with ganglioside cross-linking than with Fc RI stimulation [12]. In addition to these effects of mAbAA4, preincubation of RBL-2H3 cells with mAbAA4 selectively inhibits the degranulation induced by Fc RI stimulation at a very early step of upstream receptor tyrosine phosphorylation. This inhibition is unrelated to mAbAA4 blocking IgE-binding to the cells [48,49]. Moreover, the GD1b derived gangliosides coimmunoprecipitate with Fc RI [48] as well as with the tyrosine kinase Lyn [49]. Oliver et al. [50] demonstrated that in RBL-2H3 cells stimulated via Fc RI, the gangliosides and Fc RI are internalized together and follow the same intracellular endocytic pathway suggesting that the GD1b derived gangliosides are involved in the organization of the signaling complex.

Conclusions
The present study has demonstrated that cross-linking the GD1b derived gangliosides stimulates the release of newly formed and newly synthesized mediators. Although these gangliosides are intimately associated with Fc RI, the ability of the gangliosides to activate mast cells is not dependent on Fc RI cross-linking. The present study helps to explain the extremely broad spectrum of potential mechanisms by which mast cells might act in suppressing, amplifying, and modulating the non-Fc RI mediated immune responses. Furthermore, an understanding of the role of gangliosides in mast cell activation may lead to new therapeutic targets for allergic and inflammatory processes.