Alasdair M. Gilfillan

Integrated signalling pathways for mast-cell activation

Mast-cell activation mediated by the high-affinity receptor for IgE (FcεRI) is considered to be a key event in the allergic inflammatory response. However, in a physiological setting, other receptors, such as KIT, might also markedly influence the release of mediators by mast cells. Recent studies have provided evidence that FcεRI-dependent degranulation is regulated by two complementary signalling pathways, one of which activates phospholipase Cγ and the other of which activates phosphatidylinositol 3-kinase, using specific transmembrane and cytosolic adaptor molecules. In this Review, we discuss the evidence for these interacting pathways and describe how the capacity of KIT, and other receptors, to influence FcεRI-dependent mast-cell-mediator release might be a function of the relative abilities of these receptors to activate these alternative pathways.

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

The tyrosine kinase network regulating mast cell activation

Summary:  Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen‐mediated aggregation of immunoglobulin E (IgE)‐occupied high‐affinity receptors for IgE (FcεRI) on the mast cell surface, a response which can be further enhanced following stem cell factor‐induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both FcεRI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, FcεRI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor‐proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton’s tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain‐containing phosphatase 1 (SHP‐1) and SHP‐2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and FcεRI‐mediated mast cell activation.

Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions

BACKGROUND Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. METHODS We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. RESULTS Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. CONCLUSIONS Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Mechanisms of mast cell signaling in anaphylaxis

The recent development of a consensus definition and proposed diagnostic criteria for anaphylaxis offers promise for research efforts and a better understanding of the epidemiology and pathogenesis of this enigmatic and life-threatening disease. This review examines basic principles and recent research advances in the mechanisms of mast cell signaling believed to underlie anaphylaxis. The unfolding complexity of mast cell signaling suggests that the system is sensitive to regulation by any of several individual signaling pathways and intermediates and that complementary pathways regulate mast cell activation by amplified signals. The signaling events underlying anaphylactic reactions have largely been identified through experiments in genetically modified mice and supported by biochemical studies of mast cells derived from these mice. These studies have revealed that signaling pathways exist to both upregulate and downregulate mast cell responses. In this review we will thus describe the key molecular players in these pathways in the context of anaphylaxis.

IgE-dependent Activation of Sphingosine Kinases 1 and 2 and Secretion of Sphingosine 1-Phosphate Requires Fyn Kinase and Contributes to Mast Cell Responses

Engagement of the high affinity receptor for IgE (FcϵRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcϵRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcϵRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcϵRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.

Activation and Function of the mTORC1 Pathway in Mast Cells

Little is known about the signals downstream of PI3K which regulate mast cell homeostasis and function following FcεRI aggregation and Kit ligation. In this study, we investigated the role of the mammalian target of rapamycin complex 1 (mTORC1) pathway in these responses. In human and mouse mast cells, stimulation via FcεRI or Kit resulted in a marked PI3K-dependent activation of the mTORC1 pathway, as revealed by the wortmannin-sensitive sequential phosphorylation of tuberin, mTOR, p70S6 kinase (p70S6K), and 4E-BP1. In contrast, in human tumor mast cells, the mTORC1 pathway was constitutively activated and this was associated with markedly elevated levels of mTORC1 pathway components. Rapamycin, a specific inhibitor of mTORC1, selectively and completely blocked the FcεRI- and Kit-induced mTORC1-dependent p70S6K phosphorylation and partially blocked the 4E-BP1 phosphorylation. In parallel, although rapamycin had no effect on FcεRI-mediated degranulation or Kit-mediated cell adhesion, it inhibited cytokine production, and kit-mediated chemotaxis and cell survival. Furthermore, Rapamycin also blocked the constitutive activation of the mTORC1 pathway and inhibited cell survival of tumor mast cells. These data provide evidence that mTORC1 is a point of divergency for the PI3K-regulated downstream events of FcεRI and Kit for the selective regulation of mast cell functions. Specifically, the mTORC1 pathway may play a critical role in normal and dysregulated control of mast cell homeostasis.

5-Hydroxytryptamine Induces Mast Cell Adhesion and Migration

The neurotransmitter serotonin (5-hydroxytryptamine (5-HT)) is implicated in enhancing inflammatory reactions of skin, lung, and gastrointestinal tract. To determine whether 5-HT acts, in part, through mast cells (MC), we first established that mouse bone marrow-derived MC (mBMMC) and human CD34+-derived MC (huMC) expressed mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mouse and human MC degranulation, adhesion, and chemotaxis. We found no evidence that 5-HT degranulates MC or modulates IgE-dependent activation. 5-HT did induce mBMMC and huMC adherence to fibronectin; and immature and mature mBMMC and huMC migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin, we identified 5-HT1A as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT1A receptor knockout mouse (5-HT1AR−/−) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT1AR+/+ mice, but not in 5-HT1AR−/− mice. These studies are the first to demonstrate an effect of 5-HT on MC. Furthermore, both mouse and human MC respond to 5-HT through the 5-HT1A receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.

Btk plays a crucial role in the amplification of Fc RI-mediated mast cell activation by Kit

Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cγ1 activation and calcium mobilization, and to phosphorylate NFκB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments FcϵRI-mediated mast cell activation.

The multiple roles of phosphoinositide 3-kinase in mast cell biology

Mast cells play a central role in the initiation of inflammatory responses associated with asthma and other allergic disorders. Receptor-mediated mast cell growth, differentiation, homing to their target tissues, survival and activation are all controlled, to varying degrees, by phosphoinositide-3-kinase (PI3K)-driven pathways. It is not fully understood how such diverse responses can be differentially regulated by PI3K. However, recent studies have provided greater insight into the mechanisms that control, and those that are controlled by, different PI3K subunit isoforms in mast cells. In this review, we discuss how PI3K influences the mast cell processes described above. Furthermore, we describe how different mast cell receptors use alternative isoforms of PI3K for these functions and discuss potential downstream targets of these isoforms.