Shoko Iwaki

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.

Canonical Transient Receptor Potential 5 Channel in Conjunction with Orai1 and STIM1 Allows Sr2+ Entry, Optimal Influx of Ca2+, and Degranulation in a Rat Mast Cell Line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca2+ and the ensuing influx of external Ca2+, 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 Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca2+ and Sr2+ 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 Ca2+ but not Sr2+, whereas overexpression of STIM1 and TRPC5 allows entry of both Ca2+ and Sr2+. These and other observations suggest that the Sr2+-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ to generate a signal for degranulation.

Vascular endothelial hyperpermeability induces the clinical symptoms of Clarkson disease (the systemic capillary leak syndrome)

The systemic capillary leak syndrome (SCLS) is a rare disorder characterized by transient episodes of hypotensive shock and anasarca thought to arise from reversible microvascular barrier dysfunction. Although the high prevalence of a monoclonal gammopathy of unknown significance in SCLS suggests a pathogenic contribution of endogenous immunoglobulins, the mechanisms of vascular hyperpermeability remain obscure. Herein, we report clinical and molecular findings on 23 patients, the largest SCLS case series to date. Application of episodic SCLS sera, but neither the purified immunoglobulin fraction nor sera obtained from patients during remission, to human microvascular endothelial cells caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays without inducing endothelial apoptosis. Intravenous immunoglobulin, one promising therapy for SCLS, mitigated the permeability effects of episodic sera. Consistent with the presence of endogenous, nonimmunoglobulin, circulating permeability factor(s) constrained to SCLS episodes, we found that vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2), were elevated in episodic SCLS sera but not in remission sera. Ab-based inhibition of Ang2 counteracted permeability induced by episodic SCLS sera. Comparable experiments with anti-VEGF Ab (bevacizumab) yielded less interpretable results, probably because of endothelial toxicity of VEGF withdrawal. Our results support a model of SCLS pathogenesis in which nonimmunoglobulin humoral factors such as VEGF and Ang2 contribute to transient endothelial contraction, suggesting a molecular mechanism for this highly lethal disorder.

Amplification of Cytokine Production through Synergistic Activation of NFAT and AP-1 Following Stimulation of Mast Cells with Antigen and IL-33

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 Ca2+/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.

Generation, isolation, and maintenance of rodent mast cells and mast cell lines.

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 (FcɛRI) 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.

Antibodies to Complement Receptor 3 Treat Established Inflammation in Murine Models of Colitis and a Novel Model of Psoriasiform Dermatitis

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.

IL-33 induces a hyporesponsive phenotype in human and mouse mast cells.

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 Cγ1 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.

TLR-mediated signaling pathways circumvent the requirement for DAP12 in mast cells for the induction of inflammatory mediator release

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.

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.

Genome-wide SNP analysis of the Systemic Capillary Leak Syndrome (Clarkson disease).

The Systemic Capillary Leak Syndrome (SCLS) is an extremely rare, orphan disease that resembles, and is frequently erroneously diagnosed as, systemic anaphylaxis. The disorder is characterized by repeated, transient, and seemingly unprovoked episodes of hypotensive shock and peripheral edema due to transient endothelial hyperpermeability. SCLS is often accompanied by a monoclonal gammopathy of unknown significance (MGUS). Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. Exome capture sequencing was performed on genomic DNA from nine of these patients as validation for the SNP-chip discoveries and de novo data generation. We identified candidate susceptibility loci for SCLS, which included a region flanking CAV3 (3p25.3) as well as SNP clusters in PON1 (7q21.3), PSORS1C1 (6p21.3), and CHCHD3 (7q33). Among the most highly ranked discoveries were gene-associated SNPs in the uncharacterized LOC100130480 gene (rs6417039, rs2004296). Top case-associated SNPs were observed in BTRC (rs12355803, 3rs4436485), ARHGEF18 (rs11668246), CDH13 (rs4782779), and EDG2 (rs12552348), which encode proteins with known or suspected roles in B cell function and/or vascular integrity. 61 SNPs that were significantly associated with SCLS by microarray analysis were also detected and validated by exome deep sequencing. Functional annotation of highly ranked SNPs revealed enrichment of cell projections, cell junctions and adhesion, and molecules containing pleckstrin homology, Ras/Rho regulatory, and immunoglobulin Ig-like C2/fibronectin type III domains, all of which involve mechanistic functions that correlate with the SCLS phenotype. These results highlight SNPs with potential relevance to SCLS.