Biomedical subjects

Alasdair M Gilfillan

Publications and source records attributed to Alasdair M Gilfillan.

At least 19 recordsLinked to original sources

PubMed2006

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-HT(1A) as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT(1A) receptor knockout mouse (5-HT(1A)R(-/-)) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT(1A)R(+/+) mice, but not in 5-HT(1A)R(-/-) 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-HT(1A) receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.

Actins↗

PubMed2006

Ntal/Lab/Lat2.

Non-T cell activation linker (NTAL)/linker for activation of B cells (LAB), now officially termed LAT2 (linker for activation of T cells 2) is a 25-30kDa transmembrane adaptor protein (TRAP) associated with glycolipid-enriched membrane fractions (GEMs; lipid rafts) in specific cell types of hematopoietic lineage. Tyrosine phosphorylation of NTAL/LAB/LAT2 is induced by FcvarepsilonRI aggregation and Kit dimerization in mast cells, FcgammaRI aggregation in monocytes, and BCR aggregation in B cells. NTAL/LAB/LAT2 is also expressed in resting NK cells but, unlike the related TRAP, LAT, not in resting T cells. As demonstrated in monocytes and B cells, phosphorylated NTAL/LAB/LAT2 recruits signaling molecules such as Grb2, Gab1 and c-Cbl into receptor-signaling complexes. Although gene knock out and knock down studies have indicated that NTAL/LAB/LAT2 may function as both a positive and negative regulator of mast cell activation, its precise role in the activation of these and other hematopoietic cells remains enigmatic.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival.

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.

Calcium↗

PubMed2006

Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses.

In this article, we have described studies that have demonstrated that mast cells can be activated as a consequence of adaptive and innate immune reactions and that these responses can be modified by ligands for other receptors expressed on the surface of mast cells. These various stimuli differentially activate multiple signaling pathways within the mast cells required for the generation and/or release of inflammatory mediators. Thus, the composition of the suite of mediators released and the physiologic ramifications of these responses are dependent on the stimuli and the microenvironment in which the mast cells are activated. Knowledge of the different signaling molecules used by cell surface receptors may allow selective pharmacologic targeting such that inhibiting the adverse effects of mast cell activation can be achieved without influencing the beneficial effects of mast cell activation. The exact interconnections between the signaling pathways initiated by the surface receptors described in this article remain to be completely worked out; thus, this remains a topic for future investigation.

Animals↗

PubMed2006

Molecular regulation of mast cell activation.

The mast cell is a central player in allergy and asthma. Activation of these cells induces the release of preformed inflammatory mediators localized in specialized granules and the de novo synthesis and secretion of cytokines, chemokines, and eicosanoids. The balance of engaging inhibitory and activatory cell-surface receptors on mast cells determines whether the cell becomes active on encountering a challenge. However, recent evidence suggests that, once activated, a mast cell's response is further regulated by the balance of both positive and negative intracellular molecular events that extend well beyond the traditional role of kinases and phosphatases. These functional responses are also carefully governed by other protein and lipid mediators that determine the rate and extent of the response. Molecules that have adaptor functions, modulate lipids, and provide synergistic signals add to the regulatory complexity. Considerable information has been obtained from the study of the high-affinity receptor for IgE (FcepsilonRI), and thus it is the major focus of this review. The unifying theme is that the regulatory steps mentioned herein are required for promoting effective responses while protecting against unwanted inflammatory responses.

Animals↗

PubMed2006

Integrated signalling pathways for mast-cell activation.

Mast-cell activation mediated by the high-affinity receptor for IgE (FcepsilonRI) 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 FcepsilonRI-dependent degranulation is regulated by two complementary signalling pathways, one of which activates phospholipase Cgamma 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 FcepsilonRI-dependent mast-cell-mediator release might be a function of the relative abilities of these receptors to activate these alternative pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The high-affinity immunoglobulin-E receptor (FcepsilonRI) is endocytosed by an AP-2/clathrin-independent, dynamin-dependent mechanism.

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FepsiloncRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcepsilonRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.

Animals↗

PubMed2005

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 (FcepsilonRI) 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 FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI 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 FcepsilonRI-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.

Animals↗

PubMed2005

Btk plays a crucial role in the amplification of Fc epsilonRI-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 Cgamma1 activation and calcium mobilization, and to phosphorylate NFkappaB 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 Fc epsilonRI-mediated mast cell activation.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2005

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.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Nitric oxide inhibits IgE-dependent cytokine production and Fos and Jun activation in mast cells.

NO is a cell-derived radical reported to inhibit mast cell degranulation and subsequent allergic inflammation, although whether its action is nonspecific or occurs via specific molecular mechanisms remains unknown. To examine this question, we set out to determine whether NO inhibits mast cell cytokine production, and, if so, whether it also alters FcepsilonRI-dependent signal transduction. As hypothesized, the radical inhibited IgE/Ag-induced IL-4, IL-6, and TNF production. Although NO did not influence phosphorylated JNK, p38 MAPK, or p44/42 MAPK, it did inhibit phosphorylation of phospholipase Cgamma1 and the AP-1 transcription factor protein c-Jun, but not NF-kappaB or CREB. NO further completely abrogated IgE/Ag-induced DNA-binding activity of the nuclear AP-1 proteins Fos and Jun. These results show that NO is capable of inhibiting FcepsilonRI-dependent mast cell cytokine production at the level of gene regulation, and suggest too that NO may contribute to resolution of allergic inflammation.

Animals↗

PubMed2004

Essential role for the p110delta 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 (FcvarepsilonRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110delta 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 p110delta protects mice against anaphylactic allergic responses. These results identify p110delta as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Anaphylaxis↗

PubMed2004

Kit and FcepsilonRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells.

In human mast cells, derived from CD34(+) peripheral blood cells, we observed that Kit ligand (KL) failed to induce degranulation but acted in synergy with antigen to markedly enhance degranulation, levels of cytokine gene transcripts, and production of cytokines. Further examination revealed that antigen and KL activated common and unique signaling pathways to account for these varied responses. KL, unlike antigen, failed to activate protein kinase C but activated phospholipase Cgamma and calcium mobilization and augmented these signals as well as degranulation when added together with antigen. Both KL and antigen induced signals that are associated with cytokine production, namely phosphorylation of the mitogen-activated protein kinases, phosphatidylinositol 3-kinase-dependent phosphorylation of protein kinase B (also known as Akt), and phosphorylation of nuclear factor kappaB (NFkappaB). However, only KL stimulated phosphorylation of signal transducer and activator of transcription 5 (STAT5) and STAT6, whereas antigen weakly stimulated the protein kinase C-dependent induction and phosphorylation of c-Jun and associated activating protein-1 (AP-1) components, an action that was markedly potentiated by costimulation with KL. Interestingly, most signals were down-regulated on continuous exposure to KL but were reactivated along with cytokine gene transcription on addition of antigen. The findings, in total, indicated that a combination of FcepsilonRI and Kit-mediated signals and transcriptional processes were required for optimal physiologic responses of human mast cells to antigen.

Calcium↗

PubMed2004

Fcgamma 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 (FcepsilonRI) on the mast cell surface following antigen-dependent ligation of FcepsilonRI-bound IgE. However, data obtained from rodent and human mast cells have revealed that IgG receptors (FcgammaR) 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-gamma, FcgammaRI is upregulated, FcgammaRII is expressed but not upregulated, and FcgammaRIII is not expressed. In contrast, in mouse mast cells, FcgammaRII and FcgammaRIII receptors are expressed, whereas FcgammaRI is not. Aggregation of FcgammaRI on human mast cells promotes mediator release in a manner generally similar to that observed following FcepsilonRI aggregation. Aggregation of FcgammaRIIb in mouse mast cells fails to influence cellular processes; however, when coligated with FcepsilonRI, 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 FcgammaRs on mast cells may not be directly translatable to the human. The exploitation of FcgammaRs for a potential therapy for the treatment of allergic disorders is discussed in this context.

Amino Acid Sequence↗

PubMed2004

NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation.

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc epsilon RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc epsilon RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc epsilon RI aggregation. Fc epsilon RI- and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit- and Fc epsilon RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry.

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase.

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Comparison of Fc epsilon RI- and Fc gamma RI-mediated degranulation and TNF-alpha synthesis in human mast cells: selective utilization of phosphatidylinositol-3-kinase for Fc gamma RI-induced degranulation.

We have demonstrated that CD34(+) IFN-gamma-treated human mast cells (HuMC) express functional Fc gamma RI and that aggregation of these receptors leads to mediator release. As the signaling pathways linking Fc gamma RI aggregation to mediator release are unknown, we examined Fc gamma RI-dependent activation of specific signal transduction molecules and determined the relative involvement of these events in HuMC degranulation and TNF-alpha production following both Fc gamma RI and Fc epsilon RI aggregation. Fc gamma RI aggregation resulted in the phosphorylation/activation of src kinases and p72(syk) and subsequent tyrosine phosphorylation of multiple substrates. Inhibitor studies revealed that these responses were required for degranulation and TNF-alpha synthesis. Both Fc gamma RI and Fc epsilon RI aggregation also activated the MAP kinases ERK 1/2, JNK and p38 and this was necessary for TNF-alpha synthesis, but not degranulation for both receptors. Thus, signaling events in HuMC following aggregation of Fc gamma RI were generally similar to those observed following Fc epsilon RI aggregation. The one exception was that, although phosphatidylinositol-3-kinase was activated after both Fc epsilon RI and Fc gamma RI aggregation, only the Fc gamma RI appeared to require this molecule for degranulation.

Animals↗