[Show abstract][Hide abstract] ABSTRACT: IL-33 is elevated in afflicted tissues of patients with mast cell (MC)-dependent chronic allergic diseases. Based on its acute effects on mouse MCs, IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. In this study, we found that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to Ag. This reduction required >72 h exposure to IL-33 for onset and 1-2 wk for reversion following IL-33 removal. This hyporesponsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation, including Ag-mediated calcium mobilization and cytoskeletal reorganization, potentially as a consequence of downregulation of the expression of phospholipase Cγ(1) and Hck. These findings suggest that IL-33 may play a protective, rather than a causative, role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to downregulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.
The Journal of Immunology 12/2012; 190(2). DOI:10.4049/jimmunol.1201576 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-33 is associated with atopic and autoimmune diseases and, as reported here, it interacts synergistically with Ag to markedly enhance production of inflammatory cytokines in rodent mast cells even in the absence of degranulation. Investigation of the underlying mechanisms revealed that synergy in signaling occurred at the level of TGF-β-activated kinase 1, which was then transmitted downstream through JNK, p38 MAP kinase, and AP-1. Stimulation of the Ca(2+) /calcineurin/NFAT pathway by Ag, which IL-33 did not, was critical for the synergy between Ag and IL-33. For example, selective stimulation of the NFAT pathway by thapsigargin also markedly enhanced responses to IL-33 in a calcineurin-dependent manner. As indicated by luciferase-reporter assays, IL-33 failed to stimulate the transcriptional activities of NFAT and AP-1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NF-κB transcriptional activity by IL-33 was also essential for the synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP-1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag toward enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases.
European Journal of Immunology 03/2011; 41(3):760-72. DOI:10.1002/eji.201040718 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TLR, expressed on the surface of mast cells, respond to a variety of bacterial and viral components to induce and enhance high-affinity IgE receptor-mediated cytokine production. Recent reports have indicated that specific TLR-dependent responses in macrophages and dendritic cells are regulated by the ITAM-containing molecule, DAP12. When phosphorylated, DAP12 recruits Syk, which is a critical molecule for mast cell activation. We therefore examined whether DAP12 similarly regulates TLR-mediated responses in mast cells. DAP12 was confirmed to be expressed in both human and mouse mast cells and, upon phosphorylation, to recruit Syk. However, although TLR agonists induced cytokine production, and synergistically enhanced high-affinity IgE receptor-mediated cytokine production, surprisingly, they failed to increase DAP12 phosphorylation in mouse bone marrow-derived mast cells (BMMC). Furthermore, normal TLR-mediated responses were observed in DAP12(-/-) BMMC. However, DAP12 phosphorylation and subsequent Syk recruitment were observed in BMMC following Con A-induced aggregation of mannose-glycosylated receptors, and these responses, together with Con A-induced degranulation, were substantially reduced in the DAP12(-/-) BMMC. These data demonstrate that TLR have differential requirements for DAP12 for their function in different cell types and that the inability of TLR to influence mast cell degranulation may be linked to their inability to utilize DAP12 to recruit Syk.
European Journal of Immunology 12/2010; 40(12):3557-69. DOI:10.1002/eji.201040573 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.
The Journal of Immunology 03/2008; 180(4):2233-9. DOI:10.4049/jimmunol.180.4.2233 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An increase in cytosolic Ca2+ levels in mast cells provides an essential signal for exocytotic release of granules whether cells are stimulated via the IgE receptor (FcεRI) or thapsigargin (1, 2). The increase in cytosolic Ca2+ is associated with release from intracellular stores and influx of external Ca2+ (3, 4) by a mechanism, originally referred to as capacitative calcium entry (CCE) but now often referred to as store-operated calcium entry (SOCE), in which depletion of intracellular stores of Ca2+ leads to entry of Ca2+ (5, 6). Ca2+-entry was initially thought to occur through a calcium-release activated calcium current (ICRAC or CRAC) that was first identified in mast cells by patch-clamp techniques (7, 8). CRAC is a Ca2+-selective current of low conductance that requires external Ca2+ to achieve maximal conductance. Replacement of Ca2+ with Sr2+ or Ba2+ results in a decline in CRAC activity (9, 10). Nevertheless, it appears unlikely that CRAC is the sole mechanism for conductance of Ca2+ as early studies of calcium-dependent exocytosis clearly demonstrated that stimulated mast cells become highly permeable to a variety of divalent cations including Sr2+, Ba2+, and Mn2+ and that such ions can support degranulation in the absence of Ca2+ (11-13).Members of the subfamily of transient receptor potential (TRP) channels, especially the TRP canonical (TRPC) channels, were initially considered as candidates for mediating SOCE (14, 15). The TRPCs are activated as a consequence of inositol 1,4,5-trisphosphate generated through phospholipase C (PLC). Some TRPCs, such as TRPC1 and TRPC4, are activated by store depletion via inositol 1,4,5-trisphosphate or thapsigargin as demonstrated in cells deficient in TRPC1 or TRPC4 (16) or by overexpression (14, 15, 17-19) or knock down (18, 20-25) of these proteins. TRPC5 may be regulated by a variety of signals including Ca2+-store depletion (26) although there are contrary reports (27, 28). On the basis of structure and function, TRPC1, 4, and 5 appear to form one category of TRPCs and TRPC3, 6, and 7 another (29). The latter category appears to be activated primarily by diacylglycerides (i.e. receptor-operated) rather than store depletion (30, 31). However, TRPCs can conduct divalent cations such Sr2+ and Ba2+ in addition to Ca2+ and none of the TRPCs appear to have the exact electrophysiological features of CRAC (32).The molecular identity of CRAC has been clarified recently with the identification of two proteins, STIM1 a Ca2+sensor and Orai1 a channel protein, which are essential for CRAC activity. STIM1 was first identified as a component of CRAC in Drosophila S2 cells and Jurkat T cells by use of inhibitory RNAs (33). Of the two known mammalian homologues (STIM1 and STIM2), STIM1 appears to be the primary sensor of Ca2+ in intracellular stores in mammalian cells (33-37). STIM1 is strategically located in the endoplasmic reticulum and knock down of STIM1, but not of STIM2, by siRNA abolishes SOCE and CRAC following Ca2+-store depletion. The second component of CRAC was identified as a mammalian homologue of Drosophila Orai (38) also referred to as CRACM (39). A key discovery was the presence of an inactivating mutation of Orai1 in a patient with severe immunodeficiency (SCID) that was associated with low CRAC activity in T cells (38, 40). Subsequent studies showed that coexpression of Orai1 and STIM1 dramatically enhanced CRAC and SOCE (41-43). A paradoxical and unexplained finding was that overexpression Orai1 by itself suppresses CRAC and SOCE. In addition, the electrophysiological features of coexpressed Orai1 and STIM1 did not match those originally described for CRAC, which left the possibility that additional molecules might be involved (32). Recent work now indicates that TRPCs may associate with STIM1 and Orai1 and thus alter channel properties as well as enhance Ca2+ entry (44-46).We investigated the potential role of endogenous TRPCs in supporting entry of Ca2+ and Sr2+ as well as degranulation in a rat mast cell (RBL-2H3) line by overexpression or knock down of TRPCs, Orai1, and STIM1. As reported here, we found that, among the various TRPCs expressed in these cells, TRPC5 permitted influx of Sr2+, optimal influx of Ca2+, and degranulation. However, TRPC5 function was dependent on Orai1 and STIM to suggest that TRPC5 acted in conjunction with these two proteins.
[Show abstract][Hide abstract] ABSTRACT: Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcepsilonRI-mediated signaling would be an attractive approach for targeting mast cell-driven allergic reactions. To explore this concept, we examined the effects of hypothemycin, a molecule that we identified as having such properties, in human and mouse mast cells. Hypothemycin blocked Kit activation and Kit-mediated mast cell adhesion in a similar manner to the well characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcepsilonRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcepsilonRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle for a coordinated approach for the suppression of mast cell activation and provide a rationale for the development of compounds with a similar therapeutic profile.
Journal of Pharmacology and Experimental Therapeutics 02/2008; 324(1):128-38. DOI:10.1124/jpet.107.125237 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transmembrane adaptor protein (TRAP), NTAL, is phosphorylated in mast cells following FcvarepsilonRI aggregation whereby it cooperates with LAT to induce degranulation. The Kit ligand, stem cell factor (SCF), enhances antigen-induced degranulation and this also appears to be NTAL-dependent. However, Kit and FcvarepsilonRI appear to utilize different mechanisms to induce NTAL phosphorylation. Thus, we examined whether the responsible kinases selectively phosphorylated distinct tyrosines in NTAL and explored the implications for downstream signaling. Whereas FcvarepsilonRI required Lyn and Syk for NTAL phosphorylation, Kit appeared to directly phosphorylate NTAL. Furthermore, co-transfection studies with NTAL constructs revealed that Lyn, Syk, and Kit phosphorylate different tyrosines in NTAL. The tyrosines principally phosphorylated by Syk were recognized as Grb2-binding sites, whereas Lyn and Kit phosphorylated other tyrosines, both inside and outside of these motifs. Pull down studies revealed that PLCgamma1 associated with the two terminal Syk-phosphorylated Grb2-binding sites, which would help to explain the observed decrease in antigen-induced calcium signal and degranulation in NTAL-knock down-human mast cells. The observations reported herein support the conclusion that NTAL may be differentially utilized by specific receptors for relaying alternative signals and this suggests a flexibility in the function of TRAPs not previously appreciated.
[Show abstract][Hide abstract] ABSTRACT: 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.
The International Journal of Biochemistry & Cell Biology 02/2007; 39(5):868-73. DOI:10.1016/j.biocel.2006.10.018 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prior studies indicated the ability of Abs to complement receptor 3 (CR3, CD11b/CD18) to suppress the production of IL-12 from immune cells. Therefore, we tested the ability of an anti-CR3 Ab (clone M1/70) to treat established IL-12-dependent Th1-mediated inflammation in murine models. Systemic administration of anti-CR3 significantly ameliorated established intestinal inflammation following the intrarectal administration of trinitrobenzene sulfonic acid (TNBS-colitis), as well as colitis and skin inflammation in C57BL/10 RAG-2(-/-) mice reconstituted with CD4+CD45RBhigh T cells. The hyperproliferative skin inflammation in this novel murine model demonstrated many characteristics of human psoriasis, and was prevented by the adoptive transfer of CD45RBlow T cells. In vitro and in vivo studies suggest that anti-CR3 treatment may act, at least in part, by directly inhibiting IL-12 production by APCs. Administration of anti-CR3 may be a useful therapeutic approach to consider for the treatment of inflammatory bowel disease and psoriasis in humans.
The Journal of Immunology 12/2006; 177(10):6974-82. DOI:10.4049/jimmunol.177.10.6974 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe the protocols used for the isolation and/or culture of these cells and discuss the relative merits of their use.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 10/2006; Chapter 3:Unit 3.23. DOI:10.1002/0471142735.im0323s74
[Show abstract][Hide abstract] ABSTRACT: 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.
Immunology and Allergy Clinics of North America 09/2006; 26(3):427-50. DOI:10.1016/j.iac.2006.05.011 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Chemical immunology and allergy 02/2005; 87:43-58. DOI:10.1159/000087570
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: BackgroundSCF potentiates antigen mediated secretory responses in mast cells. We therefore explored whether the signaling pathways regulated by their respective receptors Kit and FcϵRI may share a common early event to account for these synergistic responses.