Novel approaches to the inhibition of mast cells in allergic disease

IgE-mediated allergic disease is a global health burden and major drain on healthcare resources [1, 2]. Allergic disease can present in many ways depending on the tissues affected, from acute life-threatening episodes of anaphylaxis due to food allergy, the strictly seasonal symptoms due to grass pollen-induced summer rhinoconjunctivitis (hayfever), or the chronic on-going inflammation evident in bronchial asthma. Current treatment approaches for these common diseases are often of poor efficacy or have unacceptable side-effects, and there is therefore an unmet need for novel inhibitors of allergic disease. Mast cells play a central role in the pathophysiology of allergy through their ability to release a plethora of pro-inflammatory autacoid mediators, proteases and cytokines in response to both high-affinity IgE receptor (FcɛRI)-dependent and -independent activation [3]. Within diseased tissues, mast cells relocate to key tissue structures such as the airway smooth muscle [4] and mucous glands [5] in asthma, facilitating the specific targeting of their mediators and adhesive cell–cell signals to these dysfunctional airway elements. Targeting mast cell activation therefore has the potential to attenuate allergic disease. It is beyond the scope of this article to describe in detail the signalling pathways activated in mast cells following IgE-dependent activation, but there are several recent state-of-the-art reviews by others [6, 7]. It is important to note that most of the signalling events identified following FcɛRI engagement have been defined in rodent models, with relatively little known about events in human mast cells. Where human cells have been investigated, some important differences in signalling components have been observed [8]. In brief, FcɛRI is a tetrameric structure that comprises an α-chain (FcɛRIα) that binds IgE, a β-chain signal-amplifying subunit (FcɛRIβ), and two γ-subunits that exist as a signalling homodimer (FcɛRIγ) (Fig. 1). Signalling in vivo is initiated when multivalent allergen binds to allergen-specific IgE bound to the FcɛRIα chain. This promotes FcɛRI aggregation and can be mimicked in vitro by the use of anti-IgE antibodies. Receptor aggregation initiates association of the receptor with lipid rafts containing Lyn kinase, a membrane-anchored member of the Src family of protein tyrosine kinases [9]. Lyn phosphorylates tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the β and γ-chains before binding strongly to the phosphorylated FcɛRIβ ITAM. The phosphorylated ITAMs in the γ-chains promote the docking of spleen tyrosine kinase (Syk). Lyn and Syk are themselves phosphorylated by recruited Lyn and Syk kinases, promoting the Syk activation loop which results in a fully activated Syk with adjacent tyrosine phosphorylation of LAT (linker of activation for T cells) [10, 11]. This promotes a cascade of events leading to the activation of PLCγ and subsequent generation of inositol trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP2). The generation of IP3 induces Ca2+ mobilization from intracellular stores, which initiates the influx of extracellular Ca2+ via store-operated Ca2+ channels (Fig. 1) [12]. DAG activates PKC which is required for degranulation to proceed and which contributes to gene transcription [13, 14]. In addition to the activation of the PLCγ pathway, there is also evidence for the involvement of PI-3 kinase-dependent [15], sphingosine kinase-dependent [16] and Ras-dependent signalling pathways [17, 18], which contribute to Ca2+ mobilization, arachidonic acid metabolism and gene transcription. Simplified schematic demonstrating the proximal FcɛRI-dependent signalling pathway and its link to the PLCγ and Ras-MAP kinase signalling modules (see text for details). Abbreviations: DAG, diacylglycerol; Grb2, growth factor receptor-bound protein 2; IP3, inositol-1,4,5-triphosphate; LAT, linker for activation of T cells; MAPK, mitogen-activated protein kinases; PIP2, phosphatidylinositol-4,5-biphosphate; PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; RER, rough endoplasmic reticulum; SOS, son-of-sevenless homologue. In terms of IgE-dependent mediator release in both rodents and humans, influx of extracellular Ca2+ is a critical requirement for the release of both pre-formed granule-derived mediators and newly generated autacoids (leukotriene C4 and prostaglandin D2 from membrane arachidonic acid) and many cytokines [19, 20]. The process of Ca2+ entry through the plasma membrane as a result of the depletion of Ca2+ from internal stores is termed capacitive Ca2+ entry. The Ca2+ current passing through the plasma membrane is known as the Ca2+ release activated Ca2+ current (ICRAC) [12], and is believed to play a central role in many physiologic processes such as gene transcription, proliferation and cytokine release [21]. ICRAC has been well characterized electrophysiologically in several cells including rodent mast cells [22], but the channel carrying it has only recently been identified as CRACM1 (also known as Orai1) [23–26]. In mouse mast cells at least, CRACM1 appears to make a major contribution to degranulation, and LTC4 and TNF-α release in response to IgE-dependent activation [27]. Human mast cells also express several members of the transient receptor potential family of ion channels that might also contribute to Ca2+ influx following immunologic activation [28, 29]. K+ channels are also of interest because they modulate Ca2+ influx and hence mediator release due to their profound effects on the cell membrane potential [30]. The K+ channels present in human mast cells differ from those in rodents, highlighting an example of species-dependent heterogeneity. For example, although human mast cells express Kir family mRNA [28, 31], they do not express functional Kir currents [8, 30, 32–36], in contrast to rodent mucosal mast cells [37, 38]. Following IgE-dependent activation, human mast cells rapidly open the intermediate conductance Ca2+-activated K+ channel KCa3.1 [8, 34, 36, 39], which has not been described to date in rodent mast cells. The KCa3.1 channel indirectly enhances Ca2+ influx and histamine release, but is not critical for secretion, and can thus be considered as increasing the gain of an immunologic stimulus [30]. This channel is closed by compounds that inhibit mast cell secretion such as β2-adrenoceptor agonists [34] and adenosine [36], providing a mechanism for the coupling of receptor activation and impaired secretion. Interestingly, this effect appears to act through a cAMP-independent mechanism [34]. It is important to note that mast cells have the potential to be activated through numerous FcɛRI-independent mechanisms which may play important roles in inflamed tissues. Examples include proteases including the mast cell protease tryptase [40], cytokines such as SCF, TNF-α, and IFN-γ [41–44], adenosine [45], Toll-like receptor ligands [46, 47], neuropeptides (particularly, skin mast cells) [48], complement [49] and hyperosmolality [50]. The pattern of mediators released by mast cells varies depending on the stimulus indicating the versatility of the mast cell in being able to respond to a number of diverse tissue insults with an appropriate pattern of mediator secretion and gene expression aimed at initiating an appropriate programme of inflammation and repair [51]. The signalling pathways involved in these FcɛRI-independent mechanisms of activation are poorly understood or unknown. From the above outline, it is evident that there are numerous points at which mast cell activation or its effects could be targeted. Starting proximally, these include allergen avoidance or immunotherapy, anti-IgE therapy to inhibit both allergen-dependent and monomeric IgE-dependent signalling [52, 53], inhibition of the various signalling components highlighted earlier in the text including the final effector ion channels required for normal stimulus secretion coupling, or antagonism of released mediators. A further approach might be to prevent the relocation of mast cells to key tissue structures such as the airway smooth muscle in asthma, and the CXCL10/CXCR3 axis and KCa3.1 ion channel appear to be promising targets for this [35, 54]. Allergen avoidance is difficult to achieve, desensitizing immunotherapy is effective but complicated and occasionally hazardous, while anti-IgE therapy is partially effective but prohibitively expensive in many healthcare systems. Some drugs are described as mast cell ‘stabilizers’ because they can inhibit IgE-dependent mast cell activation in vitro, but in vivo they are of poor efficacy. For example, disodium cromoglycate is only a weak inhibitor of IgE-dependent HLMC secretion with maximal inhibition of histamine release in vitro of 10–20% when used in the high micromolar range and it also exhibits rapid tachyphylaxis [55]. β2-Adrenoceptor agonists such as salbutamol are more potent inhibitors of HLMC mediator release in vitro [55], but again there is rapid tachyphylaxis, so that with chronic administration, the clinical evidence is that they do not attenuate mast cell secretion in the asthmatic airway and may even enhance it [56–58]. Specific inhibitors of mast cell-derived mediators such as antihistamines and leukotriene receptor antagonists are partially efficacious in some diseases such as rhinoconjunctivitis but not in all patients. Identifying effective inhibitors of mast cell mediator release therefore remains a major therapeutic goal. In this issue of the journal Lee et al. investigated the anti-allergic properties of several plant extracts [59]. They demonstrated that the leaf extract of Camellia japonica (LECJ) is able to markedly attenuate the IgE-dependent degranulation of rodent bone marrow-derived mast cells, rat basophilic leukaemia (RBL) cells, and human cord blood-derived mast cells, as well as markedly inhibit IL-4 and TNF-α mRNA expression and protein secretion from RBL cells. LECJ did not inhibit thapsigargin-induced degranulation, indicating that the active components were inhibiting the proximal FcɛRI-dependent signalling pathway. This was confirmed with the demonstration that LECJ inhibited Syk and LAT tyrosine phosphorylation inhibited Lyn activity in an in vitro phosphorylation assay and inhibited phosphorylation of several MAP kinases. LECJ was also active in vivo in the mouse model of passive cutaneous anaphylaxis, where it attenuated the extravasation of Evan's blue dye. HPLC identified eugenol and quercetin-3-β-d-glucoside as two components of LECJ, which were able to attenuate mast cell degranulation in the micromolar range. These results are very similar to the effects of the herb Polygoni cuspidati radix reported previously by this research group [60]. Although the active components were distinct, the P. cuspidati radix extract was very similar in potency to LECJ on a microgram basis. This study suggests that LECJ and its components have the potential to attenuate diseases, where IgE-dependent mast cell activation plays a dominant role. However, Lyn and Syk are used widely for signalling by many receptors in many cell types, so mast cell-independent effects are therefore also likely to occur. However, in inflamed tissues where many cell types are active, this may be an advantage, and so LECJ and its components might prove to be more active and effective than anti-IgE therapy, for example, which is much more mast cell-specific. While Lyn is undoubtedly an important initiator of FcɛRI-dependent signalling, knockdown/knockout experiments in mice have sometimes revealed enhanced FcɛRI-dependent mast cell activation (reviewed in Refs. [5] and [6]). This is probably because Lyn can also deliver inhibitory signals which are related to the intensity of the stimulus. As LECJ appears to act primarily on the inhibition of Lyn, its effects on FcɛRI-dependent mast cell activation in vivo may therefore not necessarily be inhibitory. A Syk inhibitor R112 demonstrated modest short-term efficacy in patients with seasonal allergic rhinitis when administered intranasally [61], but with regular dosing for 1 week this was ineffective [62], and R112 did not attenuate the nasal response to allergen challenge [63]. It will be interesting to see how LECJ compares with this. A further area that will require investigation is the ability of LECJ to inhibit IgE-independent mast cell activation. The importance of these IgE-independent pathways is probably underestimated in ‘allergic’ disease, and the signalling pathways are poorly understood. In summary, LECJ represents a novel inhibitor of mast cell degranulation and cytokine secretion in vitro, which acts on the proximal FcɛRI-dependent signalling pathway. It will be of great interest to see whether this will translate into a useful therapy for the treatment of allergic disease.