Christine Tkaczyk

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.

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.

Fcγ Receptors on Mast Cells: Activatory and Inhibitory Regulation of Mediator Release

Mast cell activation and subsequent release of proinflammatory mediators are primarily a consequence of aggregation of the high affinity receptors for IgE (FcΕRI) on the mast cell surface following antigen-dependent ligation of FcΕRI-bound IgE. However, data obtained from rodent and human mast cells have revealed that IgG receptors (FcγR) can both promote and inhibit mast cell activation. These responses appear to be species and/or mast cell phenotype dependent. In CD34+-derived human mast cells exposed to interferon-γ, FcγRI is upregulated, FcγRII is expressed but not upregulated, and FcγRIII is not expressed. In contrast, in mouse mast cells, FcγRII and FcγRIII receptors are expressed, whereas FcγRI is not. Aggregation of FcγRI on human mast cells promotes mediator release in a manner generally similar to that observed following FcΕRI aggregation. Aggregation of FcγRIIb in mouse mast cells fails to influence cellular processes; however, when coligated with FcΕRI, signaling events thus activated downregulate antigen-dependent mediator release. These divergent responses are a consequence of different motifs contained within the cytosolic tails of the signaling subunits of these receptors and the specific signaling molecules recruited by these receptors following ligation. The studies described imply that data obtained in rodent models regarding the influence of FcγRs on mast cells may not be directly translatable to the human. The exploitation of FcγRs for a potential therapy for the treatment of allergic disorders is discussed in this context.

Activation of human mast cells through the high affinity IgG receptor.

Mast cells are known to participate in the induction of inflammation through interaction of antigen with specific IgE bound to the high affinity receptor for IgE (FcepsilonRI). Human mast cells, derived from CD34(+) hematopoietic precursors, not only express FcepsilonRI but also express high affinity receptors for IgG (FcgammaRI), the latter only after IFN-gamma exposure. Human mast cells that express FcgammaRI are activated following FcgammaRI aggregation, either using antibodies directed to the receptor or through IgG bound to the receptor. This activation results in degranulation, with the release of granule-associated mediators, and the generation of metabolites of arachidonic acid and secretion of chemokines and cytokines. These observations provide evidence that human mast cells may also be recruited into inflammation through IgG-dependent mechanisms.

Determination of protein phosphorylation in FcεRI-activated human mast cells by immunoblot analysis requires protein extraction under denaturing conditions

The advent of activation state antibodies has greatly facilitated studies aimed at understanding the intracellular signaling cascade following occupancy and/or aggregation of surface receptors. As part of an ongoing study investigating the signal transduction cascade initiated following aggregation of the high affinity receptor for IgE (Fc epsilon RI) in human mast cells, we observed substantial differences in responses monitored by these antibodies when cells were extracted either under nonreducing or reducing conditions. This was true even in the presence of high concentrations of protease inhibitors. Although the activation of some proteins such as those of the MAP kinase pathway appeared to be unaffected by the extraction conditions, other signals, including overall tyrosine phosphorylation and activation of phospholipase Cgamma(1), were substantially different. This was due to the significant proteolysis in samples extracted under nondenaturing conditions. When the signaling proteins were extracted rapidly under denaturing conditions, however, there was little evidence of proteolysis of the signaling proteins with a resulting improved recovery of signal. Thus, accurate determination of signaling events utilizing activation state-specific antibodies in human mast cells requires protein extraction under denaturing conditions. The data presented in this report would be applicable to other cell types where high concentrations of proteases are present.

Comparison of FcϵRI- and FcγRI-mediated degranulation and TNF-α synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for FcγRI-induced degranulation

We have demonstrated that CD34+ IFN‐γ‐treated human mast cells (HuMC) express functional FcγRI and that aggregation of these receptors leads to mediator release. As the signaling pathways linking FcγRI aggregation to mediator release are unknown, we examined FcγRI‐dependent activation of specific signal transduction molecules and determined the relative involvement of these events in HuMC degranulation and TNF‐α production following both FcγRI and FcϵRI aggregation. FcγRI aggregation resulted in the phosphorylation/activation of srckinases and p72syk and subsequent tyrosine phosphorylation of multiple substrates. Inhibitor studies revealed that these responses were required for degranulation and TNF‐α synthesis. Both FcγRI and FcϵRI aggregation also activated the MAP kinases ERK 1/2, JNK and p38 and this was necessary for TNF‐α synthesis, but not degranulation for both receptors. Thus, signalingevents in HuMC following aggregation of FcγRI were generally similar to those observed following FcϵRI aggregation. The one exception was that, although phosphatidylinositol‐3‐kinase was activated after both FcϵRI and FcγRI aggregation, only the FcγRI appeared to require this molecule for degranulation.

Roles of Adaptor Molecules in Mast Cell Activation

The release of pro-inflammatory mediators from mast cells generally occurs following antigen-dependent aggregation of the high-affinity receptors for IgE (FcepsilonRI) expressed on the cell surface. Under the appropriate conditions, however, other receptors including the high-affinity receptor for IgG (FcgammaRI), Kit, the C3a complement component receptor, and adenosine receptors, can also induce or potentiate mast cell activation. In contrast, receptors such as the FcgammaRIIb low-affinity IgG receptor, and gp49b, when co-ligated with FcepsilonRI, down-regulate mast cell activation. The driving force by which the FcepsilonRI, the FcgammaRI, Kit, and potentially other receptors, lead to mast cell degranulation, arachidonic acid metabolism and cytokine gene expression, is a series of tyrosine kinase-mediated protein phosphorylation events which result in recruitment and subsequent activation of signaling enzymes. Similar processes are required by gp49b and FcgammaRIIb for the down-regulation of mast cell activation. The cellular localization and sequence of these events, the subsequent amplification and diversification of the signaling cascade, and potentially, the termination of these events, are regulated by an important group of signaling proteins termed adaptor molecules. In this chapter, we discuss the structure and properties of these molecules and how these proteins regulate the cellular processes associated with receptor-mediated mast cell activation.