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 .
gangliosides are present on the surface of mast cells and are critical for mast cell function .
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 ( fcri ) is the best characterized form of activation .
it occurs when multivalent antigens cross - link antigen - specific immunoglobulin e ( ige ) bound to fcri on the mast cell surface .
cross - linking fcri initiates a signal transduction cascade that is dependent on the tyrosine kinase syk .
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 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 ] .
cross - linking 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 syk - dependent .
rbl-2h3 cells , a rat mast cell line , a syk - negative variant of rbl-2h3 cells ( c4a2 ) , the stable rbl-2h3 cell lines expressing nfb - gfp reporter ( nfb2 ; ) ; nfat - gfp reporter ( vb9 ; ) ; and syk - negative cell lines expressing nfb - gfp reporter ( ic2 ; ) ; and nfat - gfp reporter ( ih10 ; ) were generously provided by dr .
reuben p. siraganian ( national institute of dental and craniofacial research , national institutes of health , bethesda , md ) .
cells were grown as monolayers at 37c in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 15% fetal calf serum , 0.434  mg / ml glutamine , and an antibiotic - antimycotic mixture containing 100  u / ml penicillin , 100  g / ml streptomycin , and 0.25  g / ml amphotericin b ( all from life technologies , gibco , carlsbad , ca ) in a humidified incubator with 5% co2 in air .
transfected cells were selected with geneticin ( 0.4  mg / ml ) ( sigma - aldrich ; st .
mouse monoclonal antibody anti - rat gd1b derived gangliosides ( mabaa4 ) were purchased from bd biosciences ( san jose , ca ) .
rabbit polyclonal antibody anti - human phospho - cpla2 ; rabbit polyclonal antibody anti - human cpla2 ; rabbit mab anti - human phospho - erk1/2 ; rabbit mab anti - rat erk1/2 ; rabbit mab anti - human phospho - jnk1/2 ; rabbit polyclonal antibody anti - human jnk1/2 ; rabbit mab anti - human phospho - p38 ; rabbit polyclonal antibody anti - human anti - p38 , and rabbit polyclonal antibody anti - human /-tubulin were purchased from cell signaling technology inc .
rabbit polyclonal antibody anti - rat cyclooxygenase-2 ( cox-2 ) , rabbit mab anti 5-lipoxygenase ( 5-lo ) , and rabbit polyclonal antibody anti - mouse lamin b1 were purchased from abcam ( cambridge , ma ) .
donkey anti - rabbit igg conjugated to horseradish peroxidase ( hrp ) ( jackson immunoresearch laboratories inc .
, west grove , pa ) was used as the secondary antibody . 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 fcri , cells were sensitized overnight ( on ) with mouse ige anti - tnp ascites fluid ( 1  :  5,000 dilution ) and then stimulated with 50  ng / ml of dnp48-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 ( pgd2 , pge2 , ltb4 , and ltc4 ) . 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 fcri independent stimulation , cells were incubated with 0.1  g / ml of calcium ionophore a23187 ( sigma - aldrich ) .
pgd2 , pge2 , ltb4 , and ltc4 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 .
nfb2 cells , vb9 cells , ic2 cells , and ih10 cells were incubated with mabaa4 , stimulated via fcri , or with calcium ionophore for 1  h ( as described in section 2.3 ) , rinsed , and cultured for an additional 5  h ( nfb 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 ) .
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 4c 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 ) .
mean optical density of the target protein was determined using imagej software ( nih ) .
results were analyzed using graphpad 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 ; p < 0.05 was considered statistical significant .
rbl-2h3 cells and c4a2 syk - negative cells were incubated with mabaa4 for either 30  min or 1  h and then rinsed and cultured for an additional 3  h to evaluate both immediate and delayed release of lipid mediators .
the cross - linking of gd1b derived gangliosides by mabaa4 induced both immediate and delayed release of pgd2 ( figures 1(a ) and 1(b ) ) and pge2 ( figures 1(c ) and 1(d ) ) by rbl-2h3 cells , but not by syk - negative c4a2 cells ( figures 1(a)1(d ) ) .
furthermore , the amount of pge2 released following ganglioside cross - linking was higher when compared to that found after fcri stimulation .
interestingly , cross - linking gd1b derived gangliosides did not induce the release of the lt , ltb4 , and ltc4 ( see supplementary figures 1(a ) and 1(b ) in supplementary material available online at http://dx.doi.org/10.1155/2016/9160540 ) .
cross - linking gd1b derived gangliosides resulted in the release of pgs but not lts .
therefore , cpla2 phosphorylation and induction of cox-2 expression , which are required for pg generation , were investigated .
an increase in cpla2 phosphorylation was observed after incubation of rbl-2h3 cells with mabaa4 for 5  min and the levels of cpla2 phosphorylation were higher than those observed in cells stimulated via fcri ( 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 .
mast cell activation via fcri leads to transcription factor activation resulting in the production and release of cytokines .
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 fcri .
map kinases are involved in signaling pathways that lead to production of newly synthesized mediators . 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 fcri , which agrees with the amount of cytokine released .
therefore , rbl-2h3 derived gfp reporter cell lines were used to assess nfb and nfat activation .
cross - linking gd1b derived gangliosides by mabaa4 induced activation of both nfb ( 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 fri .
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 [ 2226 ] .
antibodies to gangliosides have been shown to activate pkc and increase proliferation in lymphocytes [ 27 , 28 ] , stimulate calcium influx in oligodendrocytes , and induce leukocyte degranulation .
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 .
the results of the present study show that cross - linking gd1b derived gangliosides induces release of prostaglandins , but not leukotrienes .
incubation with mabaa4 stimulated cpla2 phosphorylation and incubation with mabaa4 for extended periods of time increased cox-2 expression . the first step in eicosanoid generation is ca - dependent phosphorylation of cpla2 through the map kinase pathway .
phosphorylated cpla2 translocates to cellular membranes , principally to the nuclear envelope , where arachidonic acid ( aa ) is released from membrane phospholipids by the action of cpla2 .
aa is then metabolized either by cox-2 or cyp2e1 to produce pgs such as pge2 or by 5-lo to produce lts in concert with 5-lipoxygenase - activating protein ( flap ) on the nuclear envelope [ 32 , 33 ] .
previous studies have shown that the immediate phase of pg generation ( 530  min ) requires the action of constitutively expressed cox-1 and phosphorylation of cpla2 , while the delayed phase of pg generation ( 46  h ) depends on the induced expression of cox-2 .
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 .
these events culminate with cytokine production and release and can occur even in the absence of mast cell degranulation .
a variety of studies investigating fcri independent mast cell activation also revealed that release of proinflammatory mediators can occur in the absence of degranulation [ 3537 ] .
the production of newly synthesized mast cell mediators following fcri activation relies on map kinase signaling pathways as well as on the activation of the transcription factors nfb 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 .
jnk1/2 is responsible , at least partially , for the expression and production of several cytokines , including il-6 and tnf- in mast cells .
additionally , activation of p38 map kinase was shown to stimulate il-4 production in bone marrow derived mast cells .
when mast cells are stimulated via fcri , the transcription factors nfb 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 nfb and nfat .
however , the degree of activation by ganglioside cross - linking was less than that observed by stimulation via fcri .
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 fcri .
similar results have been reported for phosphorylation of lyn , syk , plc1 , and the - and -subunits of fcri .
the lower phosphorylation of map kinase resulted in a reduction in nfb and nfat activation leading to a decrease in il-4 and tnf- release .
in fcri stimulated mast cells , activation of nfb depends on pkc activation . on the other hand , nfat is activated by calcineurin induced dephosphorylation , a ca - 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 , which could explain the reduced activation of nfb 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 nfb and nfat in response to fcri 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 fcri activation .
binding of mabaa4 to mast cells is known to stimulate protein tyrosine phosphorylation , including phosphorylation of lyn , syk , plc1 , and the - and -subunits of fcri .
however , the rate of phosphorylation of lyn , syk , and plc1 was slower with ganglioside cross - linking than with fcri stimulation .
in addition to these effects of mabaa4 , preincubation of rbl-2h3 cells with mabaa4 selectively inhibits the degranulation induced by fcri 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 fcri   as well as with the tyrosine kinase lyn .
oliver et al .   demonstrated that in rbl-2h3 cells stimulated via fcri , the gangliosides and fcri 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 .
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 fcri , the ability of the gangliosides to activate mast cells is not dependent on fcri 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 - fcri 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 .
 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-. 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 nfb 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 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions