Mechanisms controlling mast cell activation and allergic ... · MECHANISMS CONTROLLING MAST CELL ACTIVATION AND ALLERGIC RESPONSES: PROTEINS AND LIPIDS IN HARMONY VOL. 25 NUM. 2

RESUMENLos mastocitos son componentes esenciales de la inmunidad

innata y amplifican la inmunidad adaptativa. Este último papelestá mediado a través de la unión de inmunoglobulinas a susreceptores (Fc) presentes en la superficie de estas células. La pro-ducción por el sistema inmune adaptativo de inmunoglobulinaE (IgE) en respuesta a sustancias inocuas y su reconocimiento porlos receptores en los mastocitos causan la activación de estas célu-las, lo que constituye un proceso clave en el desarrollo de reac-ciones alérgicas y asma. Por lo tanto, es importante mantener laactivación de los mastocitos bajo control. Aunque es importanteque haya un equilibrio adecuado entre receptores activadores einhibidores en los mastocitos, una vez activados éstos, múlti-ples moléculas intracelulares determinan el tipo y la magnitud dela respuesta a un determinado estímulo. Estudios de las respuestasde mastocitos tras la activación del receptor de IgE realizadostanto in vivo como in vitro han identificado a dos miembros dela familia de Src cinasas, Lyn y Fyn, como moduladores intrace-lulares negativos y positivos. Esto se debe en parte al efecto deestas cinasas en los niveles intracelulares de fosfatidilinositol(3,4,5)-trifosfato (PIP3). El aumento en los niveles intracelularesde PIP3 a través de la reducción en la expresión de la fosfatasa quedegrada PIP3 (PTEN), resulta en una alteración en la homeosta-sis y activación de los mastocitos, demostrando la importancia deeste lípido. Fyn y Lyn también regulan otras cinasas de lípidos,como la esfingosina cinasa (SphK), la cual produce el mediadorlipídico esfingosina-1-fosfato (S1P). S1P regula la quimiotaxis ydegranulación de los mastocitos. Estos estudios han revelado quelas proteínas y lípidos intracelulares cooperan en la regulación delas respuestas celulares de los mastocitos y posiblemente contri-buyen a las enfermedades alérgicas.

PALABRAS CLAVE: FcεRI/ Lyn/ Fyn/ IgE/ Mastocito/ PTEN/Degranulación/ Esfingosina cinasa.

ABSTRACTMast cells are a key component of innate immunity and serve

to amplify adaptive immunity. This latter role is mediated throughthe binding of antigen-specific immunoglobulin to Fc receptorsexpressed on their cell surface. Deregulation of the adaptive res-ponse makes the mast cell a central player in allergy and asth-ma through the binding of IgE antibodies to substances that arenormally innocuous. Thus, the necessity of controlling mast cellactivation is evident. While both activating and inhibitory cellsurface receptors on mast cells are important in determining theoutcome of a mast cells encounter with a stimulus, once activa-ted, multiple intracellular molecules determine the type and extentof the mast cell response. In vitro and in vivo studies on the cou-pling of the high affinity IgE receptor (FcεRI) to mast cell effectorresponses has identified the Src family kinases Lyn and Fyn ashaving negative and positive roles in mast cell responses. Thisis in part modulated through the impact of these protein kina-ses on the cellular levels of phosphatidylinositol (3, 4, 5)-trisp-hosphate (PIP3). The crucial role for PIP3 could be demonstratedby downregulation of PTEN expression, a phosphatase that regu-lates cellular levels of PIP3, which caused increased levels of PIP3and deregulation of mast cell homeostasis and activation. Theimportance of lipid mediators in mast cell function is furtherdemonstrated by the close link between Lyn and Fyn activity andactivation of other lipid kinases, like sphingosine kinases (SphK).By producing sphingosine-1-phosphate (S1P), SphKs contributeto mast cell chemotaxis and degranulation. These studies reveala previously unrecognized cooperation of proteins and lipids thatis likely to contribute in allergic disease.

KEY WORDS: FcεRI/ Lyn/ Fyn/ IgE/ Mast cell/ PTEN/ Degra-nulation/ Sphingosine kinase.

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RevisiónInmunología

Vol. 25 / Núm 2/ Abril-Junio 2006: 85-100

Mechanisms controlling mast cell activation and allergic responses: Proteins and Lipids in harmony

J. Rivera, A. Olivera

Molecular Inflammation Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.

MECANISMOS DE ACTIVACIÓN DE LOS MASTOCITOS Y DE LAS REACCIONES ALÉRGICAS: PROTEÍNAS Y LÍPIDOS EN ARMONÍA

Recibido: 26 Mayo 2006Aceptado: 1 Junio 2006

MECHANISMS CONTROLLING MAST CELL ACTIVATION AND ALLERGIC RESPONSES: PROTEINS AND LIPIDS IN HARMONY VOL. 25 NUM. 2/ 2006

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INTRODUCTIONControl of mast cell homeostasis and activation requires

a highly coordinated molecular machinery that is capable ofdistinguishing the appropriate response to a given stimulus(1-

4). Deregulation of these molecular events may manifest inunregulated mast cell responses that may hamper the abilityof mast cells to function in host defense or could lead to disease(Fig. 1). Of particular interest to our research efforts has beenthe study of regulatory events preceding and followingcrosslinking by multivalent antigen of the IgE bound to thehigh affinity IgE receptor (FcεRI). These events are likelydeterminants of the responsiveness of a mast cell(5-10).

In the past five years, significant advances have beenmade in understanding the molecular events that occur uponengagement of FcεRI on mast cells (Fig. 2). The findingsreflect the importance of two Src family kinases, Lyn andFyn, in the initiation of downstream signaling events and aclose collaboration of proteins and lipids in exquisite (andredundant) control of mast cell homeostasis and activation,

which is key to the role of mast cells in adaptive immunity.Lyn kinase and cholesterol-enriched membrane micro-domains (lipid rafts) are crucial for FcεRI phosphorylation,which initiates mast cell activation. However, Lyn also playsa dominant role as a negative regulator of mast cell effectorresponses(5, 6, 9). In contrast, Fyn functions to positively regulatemast cell responsiveness through its role in regulating theactivation of phosphatidylinositol 3-OH kinase (PI3K)(11, 12)

and the levels of its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Fig. 2). By artificially manipulating theintracellular levels of PIP3, through downregulation of theexpression of the phosphatase and tensin homolog deletedon chromosome 10 (PTEN) this lipid second messenger(LSM) was demonstrated to function as «gatekeeper» ofmast cell activation(13). Another product of phophatidylinositolmetabolism, namely phosphatidylinositol (4,5)-bisphosphate(PIP2), is a substrate of phospholipase Cγ, which generatesinositol trisphosphate (IP3) and diacylglycerol (DAG) thatare key in activating calcium responses and the serine/threonine

Figure 1. Mast cells express the high affinity receptor for IgE (FcεRI) and participate in acquired immunity. Crosslinking of the IgE bound to the receptor by aspecific antigen triggers numerous signaling cascades that culminate in the massive secretion into the extracellular media of preformed mediators (a processcalled degranulation) within minutes after activation. This also triggers the de novo synthesis and secretion of cytokines, chemokines, growth factors and lipidmediators, which occurs at later times after activation. Because mast cells can immediately release mediators that invoke hypersensitivity, they constituteprimary effector cells in acute IgE-associated allergic reaction. Mast cells also contribute to late phase of IgE-associated allergic reactions and chronic inflammationand are important players in anti-parasite immunity, mostly by recruiting and promoting the activation of other immune cells and by orchestrating local inflammationand remodeling in the affected tissues. Mast cells are also central components of innate host defense against bacteria and participate in the initiation of acquiredimmune reactions (i.e. phagocytosis, expression and presentation of antigen) through receptors other than FcεRI (not depicted in this figure).

protein kinase C (PKC), respectively, thus regulating mastcell responsiveness. Additionally, an intimate relationshipbetween the generation of another LSM, sphingosine-1-phosphate (S1P) (an autocrine/paracrine regulator ofmast cell chemotaxis and degranulation(14)), and the FcεRI-proximal Src PTKs, Fyn and Lyn(15, 16) reflects the importanceof cooperation between the kinases and this LSM.

THE HIGH AFFINITY RECEPTOR FOR IgE (FcεRI) ANDRECEPTOR PROXIMAL SIGNALING EVENTS

Mast cells can be activated through a wide variety ofreceptors(17). However, the principal receptor involved in

allergy and asthma is FcεRI, the receptor for IgE (Fig. 1).This receptor, which is expressed on mast cells and basophils,is tetrameric(3, 18). It is comprised of an IgE-binding a chain,a membrane tetraspanning β chain that functions to amplifythe receptor signaling abilities, and a disulfide-linkedhomodimer of γ chains that provide the receptor its signalingcompetence(18). Both the β and γ chains contain immunoreceptortyrosine-based activation motifs (ITAMs), which have beendemonstrated as essential for the amplifying and signalingcompetence of immunoreceptors (Fig. 2). The ITAM ofthe FcεRIβ contains a noncanonical tyrosine residue that issituated between the two canonical tyrosines found inconventional ITAMs. FcεRI lacks intrinsic tyrosine kinase

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Figure 2. Simplified scheme of FcεRI signaling events in mast cells. Engagement of FcεRI results in inclusion in lipid rafts, phosphorylation (P) of receptor ITAMsby Lyn kinase, and activation of Syk kinase through ITAM binding. Fyn kinase is also activated and is important for phosphorylation of the adapter known asGrb-2 associated binder-like protein 2 (Gab2) and activation of phosphatidylinositol 3-OH kinase (PI3K) activity. Lyn regulates the activation of Syk andphosphorylation of several adaptor proteins called linker for activation of T cells (LAT) and Non-T cell activation linker (NTAL). These proteins function asscaffolds and organize other signaling proteins that can impact on Ras activation, PLCγ activation through the coordinated function of Gads/SLP-76/Vav1 andTec family kinases, PI3K activity, and calcium responses. PLCγ activation can also be regulated independently of LAT through a PI3K/Btk-dependent pathway.MAP kinase and transcription factor activation is dependent on LAT (and possibly NTAL) leading to mast cell cytokine production and eicosanoid productionthrough cPLA2 activation. PLCγ-generated diacylglycerol (DAG) is key for early activation of protein kinase C (PKC) and mast cell cytokine production anddegranulation. PI3K activity is also required for activation of phospholipase D (PLD) and sphingosine kinase (SphK1 or SK). SphK generates sphingosine-1-phosphate (S1P) from sphingosine (Sph), which influences calcium mobilization and mast cell effector responses through cell surface receptors for S1P (see Fig.5). The coordination of these molecular events is intrinsically regulated by both positive and negative functions of many of the components in the signaling cascade.Reprinted from the Journal of Allergy and Clinical Immunology 2006;117:1214-1225, with permission from the Academy of Allergy, Asthma, and Immunology.

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activity and phosphorylation of the tyrosine residues in theITAM motifs occurs through transphosphorylation by Lyn(19),which interacts with the β chain of the receptor. This initialphosphorylation requires liquid-ordered phase domains inthe plasma membrane, which are enriched in cholesterol,sphingolipids and other saturated phospholipids (lipidrafts), and serve to concentrate Lyn in this region of themembrane(20). At present, the data support a role for lipidrafts in maintaining the phosphorylated state of the FcεRIrather than being essential for the initiation of itsphosphorylation(7, 17, 21). Phosphorylated ITAMs can bind toa variety of proteins that can act as positive and negativeregulators of signal amplification(17). The interactions so faridentified are those with the tyrosine kinase Syk (Fig. 2), anessential kinase for the propagation of signals, and thetyrosine phosphatases SHP-1/2, although the temporalaspects of these interactions are poorly understood.

THE ARCHITECTURE OF CELL SIGNALINGDOWNSTREAM OF FcεRI

The formation of a multi-molecular signaling complex(termed signalosome) is a necessary step in regulatingdownstream cellular processes(22). These signaling complexes(Fig. 2) must be localized to specific regions within theplasma membrane (like lipid rafts) that allow interactionswith proteins or novel lipids generated by aggregation ofsurface receptors. The formation of signalosomes is coordinatedby specific proteins termed adaptor molecules (also termedlinkers or scaffolds) that function to provide docking sitesfor other signaling proteins(23). The protein-protein andprotein-lipid interactions that occur in these signalosomescan be constitutive or inducible and are mediated by specificstructural motifs in the interacting molecules.

Immediately following FcεRI stimulation, the lipid raft residenttransmembrane adaptor molecule Linker for Activation of T cells(LAT, Fig. 2) is phosphorylated by Syk in multiple tyrosine residues,becoming an anchor for the organization of signaling proteins(24). Asecond lipid raft resident adaptor termed Non-T cell Activation Linker(NTAL, Fig. 2) is also rapidly phosphorylated after engagement ofFcεRI(25). These molecules have been proposed to have both a positiveand negative regulatory control of mast cell activation and have beenthe topic of recent reviews(1, 26). Whereas it appears that LAT is crucialfor degranulation and cytokine production, the role of NTAL is notcompletely clear as it has been proposed to mediate both inhibitoryand activating functions(1). A role for NTAL in promoting mast celldegranulation was evident in studies that downregulated NTALexpression by siRNA(27). In contrast, genetic deletion of NTAL resultedin increased mast cell degranulation(28, 29). A possible competitionwith LAT for lipid raft occupancy could explain the observed results,

since in the absence of NTAL expression more LAT might partitioninto lipid rafts, augmenting mast cell responses, as LAT-deficiencymarkedly attenuates mast cell responsiveness(29).

LAT may also serve to integrate both positive and negativesignals depending on the tissue origin of the mast cell andon the stimulus(30). Upon phosphorylation by Syk, LATregulates the activation of phospholipase Cγ (PLCγ) and themobilization of calcium responses (Fig. 2). This is dependenton the ability of PLCγ to directly bind LAT (following LATphosphorylation) and also through the increased stabilityof this complex by cooperative binding of the SH2-containingleukocyte protein of 76 kDa (SLP-76), another adaptor proteinwhich acts as a linker between PLCγ1 and LAT-bound Gads(31).Mutational studies show that the multiple tyrosine residuesin LAT contribute to mast cell responsiveness, demonstratingcooperativity of these protein binding sites(32). Consistentwith this view of functional cooperativity, LAT-deficientand SLP-76 deficient mast cells also display a reduced calciumresponse, and a reduced capacity to degranulate and generatecytokines in response to antigen(33).

Other adaptor proteins are also activated and recruitedupon FcεRI stimulation. These include Shc, Grb2, MIST/Clnk,ADAP, and other proteins detailed in previous reviews(17,

23, 34). Of particular note, the adaptor Grb2-associated binder2 (Gab2, Fig. 2) has been implicated in binding the p85regulatory subunit of PI3K in mast cells and is a key adaptorin the activation of PI3K activity in various cell types(35). Thisadaptor protein appears to function downstream of Fynactivation(11); both Fyn and Gab2-deficient mast cells showeddefective PI3K activity as well as impairment of cytokineproduction and degranulation(11, 12). Recent work places Gab2downstream of Syk and suggests that Fyn regulation of Sykactivity is important for the normal phosphorylation ofGab2(36). This implicates Fyn as a regulator of Syk activity,which merits further investigation, as Syk would play adominant role in regulating adaptor function (throughphosphorylation) in mast cells.

THE INVOLVEMENT OF LIPIDS IN MAST CELLSIGNALING AND FUNCTION

In addition to the complex regulation of signals by proteinkinases, phosphatases and adaptor proteins, the coordinatedactivation of lipid kinases, phosphatases, and phospholipasesresults in the formation of lipid mediators that contributeto the intricate array of signals regulating mast cell function.As briefly mentioned above, lipid rafts may provide theenvironment where active FcεRI-induced signaling complexesoccur(37). The exact contributions of these lipid domains tothe organization of FcεRI signaling is still a matter of


investigation and has been extensively reviewed elsewhere(37-

39). Following stimulation of FcεRI, there are considerablechanges in the overall lipid composition of lipid rafts(40) andthis may play an important role in determining mast cellresponsiveness. New evidence for this view is provided byseveral recent studies. A murine model of Smith-Lemli-Opitz syndrome, a disease where the gene for 7-dehydrocholesterol reductase is mutated resulting incholesterol deficiency, demonstrated the importance of lipidraft stability in controlling mast cell sensitivity to a stimulus(7).Mast cells from these mice showed reduced levels of Lynexpression in the lipid rafts, increased Fyn kinase and Aktactivity reflecting increased PIP3 levels, and enhanceddegranulation(7). Mast cells deficient of Lyn, SHIP, or PTEN,all of which caused increased intracellular levels of PIP3,also caused enhanced mast cell degranulation(10, 13), whereasinhibition of PIP3 production caused mast cell non-responsiveness(41). The levels of diacylglycerol, a key regulatorof protein kinase C activity in mast cells(42) is also a determinantof mast cell responsiveness and changes in the concentration

of sphingolipids are now known to both positively andnegatively influence mast cell activation(4). The productionof LSMs is dependent on a variety of proteins such asphospholipases A2 (PLA2), C and D (PLD) as well as PI3Ksand sphingosine kinases (SphKs). We detail some of theevents demonstrating the close harmony of receptors, kinases,and other signaling proteins with LSMs.

Phosphatidylinositols and diacylglycerols in the regulationof signaling and responses

Regulation of phosphatidylinositol metabolism is crucialfor effective mast cell signaling and effector responsesthrough FcεRI. This cycle is regulated by both positive andnegative protein regulators such as PI3K, PIP5K, PLCγ andSHIP or PTEN, which can act to control the levels of themultiple (phosphatidyl)inositides formed in the cell(43). Herewe focus on those events known to impact mast cell activationand function.

PI(4,5)P2 acts as a signaling lipid in mast cells by directlybinding regulatory proteins and components of the

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Figure 3. Schematic representation of the dominant lipid pathways and protein partners in mast cells. After the engagement of the FcεRI both Fyn and Lyn kinasescontribute at multiple levels to regulate lipid messengers that positively or negatively influence mast cell responses. Shown are the contribution of protein kinases,which phosphorylate and modify the function of multiple proteins; adaptor proteins, which bring activator proteins and lipid enzymes together in the proximityof the membrane; and lipid enzymes, which produce or remove lipid messengers. Lipid messengers are important in the targeting, activation and regulation of thefunction of signaling proteins. All these pathways weave a selection of dynamic signals that adjust spatio-temporally to fine-tune mast cell responses.

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endocytotic/exocytotic machinery. In addition, through theaction of the phosphodiesterases phospholipase Cγ1 and Cγ2

(PLCγ1 and PLCγ2) and the lipid kinase PI3K, two majorlipid messengers are produced from PI(4,5)P2: Diacylglycerol,which is released together with a soluble messenger IP3,and PI(3,4,5)P3, produced by the phosphorylation in the 3’-position of the inositol moiety by PI3K (Fig. 3). IP3 is essentialfor calcium mobilization from intracellular stores, andcalcium and DAG are both required for the activation ofclassical PKCs, like PKCβ, whose activity has beendemonstrated as important for mast cell degranulation (Fig.2 and 3)(44). Pharmacological stimulation of mast cells withboth calcium ionophore and DAG analogs, like phorbol 12-myristate 13-acetate (PMA), results in potent degranulation,whereas DAG analogs alone are insufficient to induce thisresponse(45). Studies using BMMC derived from PKCβ, δ orε-deficient mice have demonstrated an important role forPKCβ in mast cell degranulation and cytokine production(44,

46). In contrast, deficiency in PKCδ enhanced mast celldegranulation, particularly when the cells were weaklystimulated(47). No obvious defects in development or functionwere noted for PKCε-deficient mast cells suggesting a functionthat may be redundant with other isoforms(48).

The phosphoinositide 3-kinase (PI3K) family, on theother hand, utilizes PI(4,5)P2 to generate PIP3 (Fig. 3)(43). PIP3

binds effector proteins containingt pleckstrin homology(PH), Phox homology (PX) and epsin N-terminal homology(ENTH) domains. The PLC and PI3K-regulated signalingpathways are interdependent, not only because both enzymesact on the same substrate, but because PIP3 binds the PHdomains of certain Tec family tyrosine kinases, such as BtK,localizing them to the membrane where they promotemaximal activation of PLCγ (Fig. 3 and 4). Furthermore,PI3K is also involved in the activation of PKC by activatingPI3K-dependent protein kinase (PDK)-1(49), whichphosphorylates the activation loop sites of PKC isoforms,enhancing their activity (Fig. 3). A role of PI3K in mast celldegranulation was initially demonstrated using PI3K inhibitorssuch as wortmannin or LY294002, which blocked FcεRI-mediated mast cell degranulation as well as cytokineproduction(11, 50). There are three basic classes of PI3K (I, IIand III). The class I PI3K family is subdivided into twosubclasses, IA and IB, on the basis of molecular structureand activation mechanisms. Class IA PI3Ks are heterodimerickinases consisting of a regulatory subunit (p85α, p55α, p50α,p85β, or p55γ) and a catalytic subunit (p110α, p110β, orp110δ). The p85 regulatory subunit of PI3K interacts withphosphorylated Gab2, a process that is mediated by Fynafter the engagement of FcεRI. Genetic deletion of eitherFyn or Gab2 resulted in marked decreases in PI3K activity

and inhibition of mast cell responses(11, 35), suggesting thatClass IA PI3Ks are the isoforms directly involved in FcεRIsignaling (Fig. 2). In support of a role for type IA PI3K, mastcells derived from mice expressing p110δD910A, a loss offunction allele of the PI3K isoform p110δ, showed deficientmast cell degranulation and cytokine production(41), andmice carrying this mutation, similarly to Fyn or Gab2-deficientmice, had diminished anaphylactic reactions. This remarkablesimilarity between Fyn -, Gab2-deficient and p110δ mutantmice, suggests the possibility that p110δ is the key PI3Kisoform functioning downstream of Fyn and Gab2. Surprisingly,mast cells from mice lacking the p85 subunit of PI3K, themost abundant of the regulatory type IA class, have intactdegranulation and cytokine responses induced upon FcεRIstimulation. These mice exhibit normal passive systemicanaphylaxis as well(51). This suggests redundant functionfor some PI3K isoforms. Interestingly, class IB PI3K p110γ,which lacks the p85-binding domain, but is recruited toG-protein coupled receptors, was shown to play a role inIgE-mediated anaphylaxis and mast cell degranulation(52).The activation of p110γ did not appear to occur directlydownstream of the FcεRI, but could involve autocrine loopsof G-protein coupled receptor agonists.

The relevance of the regulation of the levels of PIP3 onmast cell responsiveness is further highlighted in studiesperformed in mast cells derived from the lipid phosphataseSHIP1-deficient mice or after downregulation of the expressionof the lipid phosphatase PTEN(10, 13) (Fig. 4). In either case,the levels of PIP3 were elevated and rendered mast cellshyperreactive. SHIP-1 regulates the FcεRI-dependentproduction of PIP3 by dephosphorylating the 5’ position togenerate PI (3,4)-P2, whereas PTEN opposes PI3K functionby dephosphorylating the 3’ position of PIP3, yielding PI(4, 5)-P2. In both SHIP-1- and PTEN-deficient mast cells,FcεRI-dependent calcium mobilization and degranulationwere enhanced. However, the respective phenotypes differedin that PTEN-deficiency caused a constitutive phosphorylationof Akt in human mast cells(13). The increased Aktphosphorylation was associated with constitutive activationof the MAP kinase family members, c-jun N-terminal kinase(JNK) and p38, phosphorylation of the transcription factorATF2 as well as IL-8 and GM-CSF secretion. This differedfrom SHIP-null murine bone marrow-derived mast cells,where resting cells showed minimal phosphorylation ofAkt and constitutive secretion of cytokines was not observed(10).Thus, Akt, JNK, and p38 MAPK appear to be highly dependenton PIP3 production for their activation and can drive theproduction and secretion of some cytokines independentlyof FcεRI engagement. Lyn is required for phosphorylationof SHIP-1(9, 53), an event important for appropriate targeting


of this lipid phosphatase (Fig. 4). Thus, in the absence ofLyn(9), or when Lyn-FcεRI interaction was impaired(53), thephosphorylation of SHIP-1 was found to be defective andPIP3 levels were increased as demonstrated by directmeasurement or indirectly by activation of Akt. This suggeststhe possibility that PTEN is required for control of PI3K inmast cell homeostasis and activation, whereas SHIP-1 isprimarily active when mast cells are activated throughFcεRI.

Sphingosine-1-phosphate; a pleiotropic regulator of mastcell function and possible allergic mediator

Metabolites of sphingomyelin, including ceramide (Cer),sphingosine (Sph), and sphingosine-1-phosphate (S1P) arealso bioactive lipids, that mediate broad-range cellularresponses such as cell growth, survival, differentiation,calcium homeostasis and chemotactic motility (Fig. 2 and4)(4, 54). S1P is involved in the positive and Sph and Cer inthe negative regulation of mast cell responsiveness(4, 55).Notably, S1P is rapidly produced after crosslinking of theFcεRI by two mammalian sphingosine kinases (SphK1 and

SphK2). Inhibition of SphK activation, and thus S1P generation,by either competitive analogs of Sph in the RBL tumor mastcell line(56) or by antisense SphK mRNA in human mastcells(57) prevented IgE-triggered calcium responses andinhibited degranulation. This was independent of Sykphosphorylation or inositol 3, 4, 5-trisphosphate (IP3)production. The involvement of S1P in calcium release bya PLCγ-independent route implies its direct or indirectaction on an unidentified channel, as has been suggestedfor other cell types(58). S1P is a ligand for a family of five G-protein coupled receptors, termed sphingosine-1-phosphatereceptors (S1P1-5) (Fig. 5)(4). Each of these S1PRs couples toa different subunit of heterotrimeric G proteins αi, αq,and α12/13, and therefore they trigger an array of signalingpathways, including activation of Src kinases, small GTPases,MAPK cascades, phopholipases, PKC and calciummobilization(59). The role of these receptors is varied, butconsiderable attention has been placed on the role of S1P1

because of its requirement for thymocyte emigration andlymphocyte recirculation(60) as well as for vascularmorphogenesis(61).

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Figure 4. Interdependence of signaling proteins and lipid messengers in mast cells. Early signaling proteins (Fyn, Lyn, and Syk) are key elements in the activationof the lipid enzymes that produce lipid second messengers (LSMs) such as phosphoinositides (PI), phosphatidic acid (PA), diacylglycerol (DAG), sphinosine(Sph) and sphingosine-1-phosphate (S1P). These LSMs can then influence the activity of the lipid enzymes (such as sphingosine kinases (SphKs)) or other signalingproteins (like PI3K-dependent protein kinase (PI3K), Bruton’s tyrosine kinase (BTK) and others). This functions to sustain and amplify the signaling reponsesthat are key to the mast cells ability to degranulate and secrete cytokines and eicosanoids.

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Mast cells express two of the five receptors for S1P, S1P1

and S1P2 (Fig. 5), and FcεRI-induced S1P formation resultsin the transactivation of these receptors(14), which in turnaffects mast cell responses. Transactivation of S1P1 is requiredfor the migration of mast cells towards low concentrationsof antigen, whereas S1P2 transactivation is important fordegranulation (Fig. 5). S1P1 may also be negatively regulatedby S1P2, since overexpression of S1P2 inhibited mast cellchemotactic motility. Interestingly, while S1P1 mRNAexpression is constitutive, S1P2 mRNA expression is enhancedas a late consequence of FcεRI crosslinking. Thus, it is possiblethat low antigen concentrations can attract mast cells to thesite of action via S1P1 and, as mast cells approach higherconcentrations of antigen, a shift in the expression of S1Preceptors (enhanced S1P2 expression) resolves migrationwhile promoting degranulation, the latter being a processknown to require a stronger stimulus(62).

The mechanism(s) by which SphKs are activated involvesa complex interplay of protein kinase- and lipid-derived

signals. SphKs translocate rapidly to plasma membranesupon FcεRI engagement and the S1P is probably generatedin the proximity of S1P receptors, resulting in their timelyactivation. The translocation of SphKs requires both Fynand Lyn (Fig. 2 and 5). Fyn activity is essential for activationof SphKs by FcεRI whereas Lyn is partly dispensable(16).Fyn-dependent activation of SphK1 is through the Gab2/PI3Kpathway outlined above(16) and includes PLD activity. Thisis consistent with the findings that phosphatidylinositols(including PIP3) and phosphatidic acid (PA) can either bind,activate or induce the translocation of SphKs(57, 63, 64). SphK2activation also required Fyn but was independent ofGab2/PI3K signals(16). These findings reveal a bifurcationof Fyn-dependent signals that was previously unappreciated.The interdependence of the SphK/S1P/S1PR axis and FcεRIproximal signaling is further highlighted by the directinteractions of Fyn and Lyn with SphKs, the dependenceon Fyn and Lyn for translocation/activation of SphKs, andthe defective degranulation and chemotaxis of Fyn-deficient

Figure 5. Role of S1P and its receptors in mast cell functions. Crosslinking of the FcεRI by IgE/antigen in mast cells results in the rapid activation and translocationof SphK to the plasma membrane and the generation of S1P. S1P, independently of PLCγ activation and IP3-generation, is thought to mobilize calcium from intracellularstores, which is necessary for mast cell degranulation. However, the nature of the intracellular calcium pools targeted by S1P has not been determined, although inthis figure it is illustrated as an ER calcium pool. S1P is secreted by activated mast cells to the extracellular media by mechanisms that have not yet been elucidated.Furthermore, generated S1P is able to rapidly bind and activate its receptors S1P1 and S1P2 on the plasma membrane. S1P1 induces cytoskeletal rearrangementsleading to the movement of mast cells towards an antigen gradient, while transactivation of S1P2 enhances the degranulation response. Mast cell secreted S1P canalso promote inflammation by activating and recruiting other immune cells involved in allergic and inflammatory responses. Since S1P also profoundly affectsendothelial cell function, induces contraction and proliferation of airway smooth muscle cells, and its levels are elevated in the bronchial lavage of asthmatic individualsafter antigen challenge, secretion of S1P by mast cells could be of relevance in this pathology. Mast cell granules are illustrated as black circles and the process ofdegranulation as granules get in contact with the plasma membrane emptying their content (smaller black dots). The thick, solid black arrow represents the intracellularactions of S1P, the dotted arrows represent the release of S1P to the extracellular media, and the dashed arrows, the signaling pathways activated via S1P receptors.Reprinted from the Journal of Immunology 2005;174:1153-1158.Copyright The American Association of Immunologist, Inc.

mast cells, which was partially corrected by the addition ofexogenous S1P upon Ag stimulation(15, 16).

Notably, S1P is one of the mediators that mast cellssecrete upon activation via antigen-specific crosslinking ofFcεRI (Fig. 5)(4). Unlike many other cells (T cells, B cells, etc.),mast cells secrete a substantial amount of S1P suggestingthat it is an important mast cell mediator. This is furthersupported by the finding that S1P was highly elevated inthe airways of asthmatic individuals(8), and in the joints ofarthritic individuals(65). Both asthma and rheumathoidarthritis are inflammatory conditions in which mast cellshave been demonstrated to be important effector cells, thelatter primarily in a mouse model(66, 67). The fact that S1Preceptors are ubiquitously expressed in a variety of celltypes and that S1P dramatically alters the function ofendothelial, epithelial and smooth muscle cells, raises thepossibility that this LSM is also a paracrine mediator involvedin the pathophysiology of asthma or other allergic and/orinflammatory diseases (Fig. 5).

Phosphatidic acid and mast cell degranulationPLD is a phosphodiesterase that hydrolyzes membrane

phosphatidylcholine (PC) to generate PA and free choline(Fig. 2-4)(68). PA is involved in a wide range of physiologicalprocesses including cytoskeletal rearrangements, vesicletrafficking, exocytosis and proliferation. The evidence forthe involvement of PA in cellular processes comes mostlyfrom studies exploiting the unique ability of the PLD enzymesto catalyze a transphosphatidylation reaction in the presenceof primary alcohols, generating, instead of PA,phosphatidylalcohols, which are presumed to be functionallyinactive. To date, two mammalian PLD isoenzymes, PLD1

and PLD2 have been cloned(68). Both isoenzymes are membrane-associated in mast cells: PLD1 is localized to perinuclearendosomes and trans-Golgi sites and PLD2 is foundpredominantly at the plasma membrane(69). Both isoformsare activated upon antigen stimulation(70) and a role for PAin degranulation has been established by the use of primaryalcohols and siRNAs directed against PLD1 and PLD2. Furtherevidence for a role of these enzymes in mast cell degranulationis provided by experiments demonstrating that expressionof a catalytically inactive mutant of PLD1 or PLD2 in mastcells blocked the migration of granules to the cell peripheryand degranulation(69, 71). Other studies have involved PLD1

in the regulation of the basal levels of PA in mast cells. Adecrease in basal levels of PA was correlated with an increasedfraction of FcεRI in lipid rafts and enhanced degranulation(72).The precise mechanism by which PLD affects degranulationis not known. Formation of PA, a cone-shaped lipid, at theexpense of lysophosphatidic acid, an inverted-cone-shaped

lipid, was found to participate in the pinching off of membranesduring endocytosis by inducing a negative membranecurvature (73). Thus, regulation of PA may alter membraneproperties facilitating membrane fusion in mast cells. Otherpossibilities include: direct signaling by PA, signaling throughits metabolic conversion to DAG mediated by PAphosphohydrolase (PAP) or through conversion tolysophosphatidic acid (another lipid messenger) mediatedby PLA2. PA, as other acidic phospholipids, can bind protein’sregulatory domains affecting their activity and ultimatelycell functions. PA was shown to activate PKCs(74), SphK(16,75),and type I PIP 5-kinase, which regulates PIP2 levels(76),and the GTPase activating proteins (GAPs) that regulatesmall GTPases(77). DAG derived from PC has also beenproposed to regulate PKC activity (Fig. 4) analogous to DAGderived from PIP2. Inhibition of PLD by 1-butanol but nottertiary-butanol inhibited the PKC translocation to themembrane fraction normally induced by FcεRI. Similarly,transfection with siRNA for PLD1 or PLD2 blocked translocationof DAG-dependent forms of PKC(78).

Although multiple pathways can regulate PLD activation,the precise mechanism for its activation via FcεRI is notknown. Both Fyn and Fgr could induce PLD2 phosphorylation,a process that correlates with PLD activation(79). Syk activityand other signals such as calcium, PKC and PI3K(80) havealso been implicated in PLD stimulation. Additionally, invitro studies suggest that PIP2 is an allosteric regulator ofboth PLD1 and PLD2 and receptor-mediated regulation ofPLD seems to be partly mediated by the small GTPases ARFand Rac1(70, 80). Collectively, the data are consistent withmultiple inputs for PLD activation and with the conceptof feedback regulatory control, since many of the signalsrequired for PLD activation (such as PKC activity) are inturn regulated by PLD activity. This may reflect the importanceof these steps in eliciting and governing mast cell function.

PROTEINS AND LIPIDS IN THE REGULATION OFTRANSCRIPTION

In addition to the FcεRI-mediated degranulation of mastcells, which releases pre-formed granule-stored mediators,stimulation of these cells leads to the secretion of de novosynthesized lipid mediators such as leukotrienes, prostaglandinsand platelet activating factor (PAF). A wide array of cytokines(IL-1-IL-6, IL-9–IL-14, IL-16, IL-18, MIF, TNF, GM-CSF, LIF,etc) and chemokines (IL-8, MCP-1–MCP-4, MIP-1, MIP-3,RANTES, etc) can also be produced and secreted (Fig.1)(2). Signals triggered by FcεRI are sufficient to activate mostenzymes in the synthetic pathway of lipid mediators leadingto their formation, whereas the de novo production of cytokines

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and chemokines requires gene transcription. The pattern ofexpression of cytokines depends on the strength of thestimulus(62) and the environment mast cells (i.e. presence ofadditional signals).

The assembly of transcription factors and DNA-bindingproteins onto specific DNA regulatory regions controlsgene transcription of cytokines or chemokines. Transcriptionfactors are modified by multiple signals and translocate toor are present in the nucleus where they interact with thepromoter regions of genes(81). Multiple transcription factorsare known to regulate cytokine expression in mast cells.Among these, nuclear factor κB (NFκB) and the nuclearfactor of activated T cells (NFAT) play major roles in theproduction of a number of cytokines by mast cells(82-86). TheNFAT family of transcription factors is known to bindcooperatively with other transcription factors like AP-1, tocomposite DNA sites. AP-1 is a hetero- or homodimericcomplex composed of members of the Fos and/or Junfamilies of transcription factors(87). In mast cells, as wellas in T cells, the NFAT complex contains Fos and Jun proteins.Depending on whether or not both transcription factorsare concomitantly activated, distinct sets of genes may beactivated, eliciting different patterns of cytokines. The majorpathways for activation of the NFAT and AP-1 transcriptionfactors are distinct, although crosstalk may occur(88). AP-1activation is regulated at multiple levels by activation ofMAP kinases, although in mast cells PKC has also beenimplicated(89). In contrast, a dominant determinant of NFATbinding activity in the nucleus is calcium mobilization(90).Jun proteins are phosphorylated by MAP kinases (e.g. JNK-1/2, Erk-1/2), while the activity of Fos family members isregulated by a rapid induction of their de novo synthesis,which appears to be regulated by PKCβ and ε in mast cells(91).Both the increased synthesis of Fos and the phosphorylationof Jun lead to the full activation of the AP-1 complex(87).Another possible partner of c-Jun in the AP-1 complex isactivating transcription factor 2 (ATF-2)(92), which is alsoregulated by MAP kinases and as indicated above by theintracellular levels of PIP3.

The MAP kinase family in regulation of mast cell geneexpression

MAP kinases play prominent roles in the transcriptionalregulation of activated mast cells (Fig. 2). The family ofMAP kinases include extracellular signal regulated kinase(ERK), c-Jun N-terminal kinase (JNK), p38, and extracellularsignal regulated kinase-5 [ERK5; also called Big MAP kinase-1 (BMK1)](93). These kinases have to some extent overlappingsubstrate specificities(94). MAP kinases regulate NFκB, NFATand AP-1 complexes, they phosphorylate and activate the

immediate early gene regulator, Elk-1(82), and their translocationinto the nucleus results in phosphorylation of other kinasessuch as the ribosomal S6 kinase (RSK) and the mitogen-and stress-activated protein kinase (MSK-1) both of whichcontribute to gene expression by phosphorylating histonesand regulatory proteins. MAP kinases are activated by dualphosphorylation in serine/threonine and tyrosine mediatedby a MAP kinase kinase (MKK), whose activation is dependenton phosphorylation by a MAP kinase kinase kinase (MKKK)(Fig. 2)(95). The most well studied of the family membersis ERK, whose activation is primarily dependent on MKKand MKK2. In contrast, JNK phosphorylation is dependenton MKK4 and MKK7 whereas p38 MAP kinase is normallya target of MKK3, MKK4, and MKK6(96). In mast cells, theparadigm of the above required hierarchy and specificityis seemingly maintained. A key component is the adaptorprotein LAT because it provides the link for activation ofthe small monomeric GTP binding protein Ras (Fig. 2).Activated Ras initiates the MAP kinase cascade by thephosphorylation of Raf (a MKKK). Deficiency in LAT or ina LAT-associated adaptor termed SLP-76 causes a markeddeficiency in activation of ERK and JNK(24, 97). NTAL hasalso been shown to contribute to MAP kinase activation(Fig. 2) but this is less well understood. Vav1, which is partof the LAT scaffold complex, is also involved in the activationof JNK, as Vav1-deficient mast cells revealed a defect in itsactivation(98). In contrast, a mast cell deficiency in NTALcaused increased activation of ERK, JNK, and p38MAPkinase suggesting that NTAL may control the extent of theiractivation(28, 29). However, mast cells deficient in both LATand NTAL showed a more marked defect than LAT-deficientmast cells in the activation of all MAP kinases(29). Thissuggests that the role of NTAL in MAP kinase regulationmight be both positive and negative. Importantly, a similardichotomy has been observed in Lyn-deficient mast cellswhere ERK activation appeared relatively normal evenwhen LAT phosphorylation was substantially reduced(11).This suggests that other pathways (possibly NTAL-mediated)are able to compensate for the absence of Lyn and thereduced LAT phosphorylation. In contrast, Fyn-deficiencyhad no effect on ERK phosphorylation but instead causeda defect in the activation of JNK and p38 MAP kinase(99).This is consistent with the aforementioned role of PIP3 inregulating the activation of these two MAP kinases asobserved by downregulation of PTEN in human mast cells(13).However, because in mast cells JNK activation is affectedby both Fyn and LAT deficiencies, crosstalk in the signalsgenerated by these proteins must occur. In support ofthis view, both Fyn- and LAT-deficient mast cells showeddefective production of IL-6 and TNF(24, 99), suggesting that


they may be important in inducing signals required fortranscription of these genes.

NFκB; an essential regulatory of proinflammatory cytokinesin mast cells

NFκB is an essential regulator of IL-6 and TNF productionin mast cells(85, 86). NFκB proteins include NFκB1 (p50and its precursor p105), NFκB2 (p52 and its precursorp100), RelA/p65, RelB, and cRel(100). The predominant formof NFκB in many cell types is a p65:p50 heterodimer,however in mast cells the predominant forms are notknown. NFκB is sequestered in the cell cytoplasm in a formbound to a family of inhibitory proteins (inhibitor of κB(IκBa), IκBβ, and IκBγ) that mask its nuclear localization

signal (Fig. 6). Phosphorylation and ubiquitination ofIκB lead to its degradation, allowing NFκB to translocateto the nucleus and to bind DNA thus promoting geneexpression. Central to the phosphorylation of IκB is theIκB kinase (IKK) complex (Fig. 6). The IKK complex, whichincludes two catalytic subunits, IKKα (or IKK1), and IKKβ(or IKK2), and a regulatory subunit, IKKγ (or NEMO)(101),can also be activated by a variety of signals to induce IκBphosphorylation. Among these, the MAP kinases, PI3K,and PKC seem dominant in many cell types. In mast cells,the mechanism by which FcεRI induces NFκB activationis still unclear, probably because of the redundancy ofsignals ensuring the activation of this central transcriptionfactor.

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Figure 6. A hypothetical model of FcεRI induction of NFkB illustrating signal divergence and selective induction of cytokine responses. FcεRI engagement resultsin the activation of protein tyrosine kinases (Src and Syk) and in calcium mobilization (Ca2+) required for activation of the conventional (calcium binding) PKCisoforms, like PKCβ. Divergence of signals likely occurs at this point through activation of various PKC isoforms and/or phosphorylation of multiple substrates.PKCβwas demonstrated to participate in mast cell degranulation. PKC-dependent activation of cPLA2, leading to eicosanoid production has also been demonstrated,although the connection to MAP kinase (MAPK) activation is not well defined (?) and alternate PKC-independent routes have also been described. CARMA1 canbe phosphorylated by PKCβ in B cells but it is not known (?) whether in mast cells this isoform is responsible for CARMA 1 phosphorylation. Once phosphorylatedCARMA1 can recruit Bcl10 and Malt1 leading to activation of the IkB kinase (IKK) complex (α, β, γ). However, the molecular connection from the CARMA1-Bcl10-Malt1 complex to IKK is not known. Phosphorylation of IkBα by IKK results in its ubiquitination and degradation, releasing active NFκB and allowingits translocation to the nucleus resulting in induction of IL-6 and TNF gene transcription. Reprinted from Trends Immunology 2006;27:251-253, with permissionof Elsevier Press.

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Nonetheless, some clues are emerging as phosphorylationof IKK was defective in Fyn-deficient mast cells(99) andthis defect resembles the defective IKK activation and NFκBactivity in Src PTK triple-deficient (Blk, Fyn, and Lyn) Bcells(102). In contrast, disruption of the interaction betweenFcεRI with Lyn (through mutation of the receptors β subunitITAM) caused enhanced IKK phosphorylation and enhancedcytokine production(53). The link between Fyn or Lyn andNFkB activation is not clear, but the data supports a modelwhereby Fyn is required for normal activation of this pathwayand receptor-associated Lyn controls the extent of IKKactivation. The role of LAT or NTAL in mast cell NFκBactivation is also unclear, however, given the defectiveproduction of IL-6 and TNF in LAT-deficient mast cells itis likely that NFκB activity is diminished by the absenceof LAT. Proteins that may be likely candidates in regulatingNFκB downstream of Fyn and Lyn include JNK, PI3K andPKC. In mast cells, these signaling proteins require Fyn fortheir full activation(11, 99) and they are crucial for NFκBactivation in various cell types. Of particular interest arethe PKCβ and θ isoforms, which play a major role in IKKphosphorylation and in cytokine production in B and T cells,respectively(103, 104). The mechanism for their key role in NFκBactivation was recently defined(105). These PKC isoforms arein the phosphorylation of an adaptor protein CARMA1important (caspase recruitment domain (CARD)-containingmember of the membrane associated guanylate cyclase(MAGUK) family-1) that scaffolds a complex of proteinsthat lead to IKK activation (Fig. 6). Once phosphorylated,CARMA1 interacts with an adaptor protein Bcl10 throughits CARD domain engendering an oligomerization that isessential for activation of IKK once Bcl10 associates with athird adaptor protein, Malt1. This complex can now causeactivation of IKK, however, the exact mechanisms by whichthis happens is not known. In mast cells (Fig. 6), it wasrecently demonstrated that Bcl10 and Malt1 are both neededfor the activation of NFκB and for IL-6 and TNF production(106).Interestingly, deficiency in either of these proteins had nosubstantial impact on mast cell degranulation or eicosanoidproduction demonstrating that the bifurcation of theseresponses from cytokine production occurs upstream ofthese two proteins(107). At the moment it is unclear if CARMA1is expressed in mast cells. However, based on the demonstratedrequirement for a scaffolding protein for Bcl10 oligomerizationand Malt1 function it is likely that a member of the CARMAfamily of proteins plays such role in mast cells(107).

Summary and PerspectivesThe findings summarized herein demonstrate the existence

of multiple signaling pathways regulating FcεRI-dependent

mast cell responses. A key feature is the complex relationshipof signaling proteins and their lipid partners, which areimportant in targeting, activating, and regulating the functionof these proteins. Several lessons can be learned from thesestudies: 1. Some lipids can directly modulate the activity ofproteins independently of a need for complementary signalsgenerated through receptor engagement. An excellentexample is provided by the findings on the shRNA silencingof PTEN in human mast cells, which demonstrated thatincreased PIP3 alone can activate Akt and MAP kinasesresulting in mast cell cytokine secretion. 2. We have alsolearned that lipid-mediated regulation occurs by modulatingintracellular signaling pathways as well as by engagementof cell surface receptors. An example of this is providedby studies on SphKs whose function in mast cells is dependenton the production of S1P that causes intracellular effects(such as calcium regulation) but also engages the S1P receptorsexpressed on mast cells to modulate both chemotaxis anddegranulation. 3. Finally, although the data is far fromdefinitive, we also learn that PLD production of PA may beessential in regulating the function of key proteins requiredfor mast cell degranulation (such as PKC). PLD productionof PA may also impact the ability of granules to fuse withmembranes, thus providing regulation for a key signal aswell as the actual process of exocytosis (upstream anddownstream regulation). Interestingly, the findings demonstratethat this protein-lipid harmony extends well beyond themast cell degranulation response alone, since cytokine geneexpression is also influenced by this interplay.

This protein-lipid mode of regulating FcεRI-dependentmast cell effector responses is not limited to these cells but isa fundamental process by which all cells are likely to beregulated in response to the surrounding stimuli. However,understanding these relationships in the mast cell has revealedseveral unique features, which suggests that several steps inthis process may be considered as possible therapeutic targetsin mast cell-related diseases like allergy. For example, therelationship between p110δ and the Fyn-Gab2 complex needsto be further explored as the data strongly suggest that thismodule may be a key component for mast cell responsesdownstream of FcεRI engagement. In particular, if FcεRI-associated Fyn is responsible for driving the Gab2-PI3K(p110δ),disruption of this interaction may be beneficial and achievablethrough targeting of receptor sequences required for theassociation of Fyn. Because FcεRI is primarily a mast cell andbasophil specific receptor, its therapeutic targeting achievesthe desired selectivity and avoids the undesired consequencesof targeting Fyn kinase activity, an enzyme expressed in avariety of cell types. Alternatively, the targeting of p110δinteractions, or its activity, may provide the needed specificity


since this kinase isoform is not expressed in all cell types.Regardless, the in-depth analysis of the mechanisms underlyingFcεRI-mediated mast cell activation are likely to provide newinsights on the pathophysiological aspects in disease. Examplesof this are provided by in vitro findings that show that Lyn-deficient mast cells are hyperactive(6). Similarly, in vivo datashow that Lyn-deficient mice develop an atopic allergic-likedisease and demonstrate an exacerbated asthmatic response(5).Given that Lyn is the enzyme required for FcεRI phosphorylation(the initiating step in IgE-dependent mast cell activation),these results were unexpected and once again teach us theunpredictability of a biological system. It is essential thatfuture efforts focus on moving the knowledge gained in vitroto suitable in vivo models that offer the opportunity to determineif the in situ milieu (both proteins and lipids) is a determinantin the observed response.

ACKNOWLEDGEMENTSMuch of the research reported herein was supported by

the Intramural Research Program of the National Instituteof Arthritis and Musculoskeletal and Skin Diseases of theNational Institutes of Health.

CORRESPONDENCE TO: Juan RiveraNIAMS/NIH, Building 10, Room 9N228Bethesda, MD, 20892-1820. Phone: 301-496-7592. Fax: 301-480-1580 Email: [email protected]

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Mechanisms controlling mast cell activation and allergic ... · MECHANISMS CONTROLLING MAST CELL ACTIVATION AND ALLERGIC RESPONSES: PROTEINS AND LIPIDS IN HARMONY VOL. 25 NUM. 2