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 C␥ 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 B (NFB). 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 Fc⑀RI and Kit-mediated signals and transcriptional processes were required for optimal physiologic responses of human mast cells to

Mast cells mediate both IgE-dependent allergic reactions and protective responses against acute infections, possibly through the activation of Toll-like receptors (TLRs). We find that antigen interacts synergistically with TLR2 and TLR4 ligands to markedly enhance production of cytokines in murine mast cell lines. However, the TLR ligands neither stimulated degranulation and release of arachidonic acid nor influenced such responses to antigen, probably because these ligands failed to generate a necessary calcium signal. The enhanced cytokine production could be attributed to synergistic activation of mitogen-activated protein kinases in addition to the engagement of a more effective repertoire of IntroductionImmunoglobulin E (IgE)-dependent atopic diseases such as allergic rhinitis, asthma, and anaphylactic reactions affect, respectively, 20%, 7%, and 2% of the population in the United States. 1,2 The respiratory manifestations of these diseases may be exacerbated by acute respiratory infections from influenza, rhino-and syncytial viruses in humans 3,4 and animals, 5,6 as well as by lipopolysaccharide (LPS), which is a ubiquitous environmental contaminant from Gram-negative bacteria. 7,8 These pathogens may act through Tolllike receptors (TLRs). 9,10 TLR4 appears to play an essential role in the immune response to respiratory syncytial virus in the human lung, 11,12 and ligands for TLR2 and TLR4 augment inflammatory responses to inhaled antigen in the mouse lung. 13,14 Thirteen TLRs have been described to date, each of which recognize specific pathogen-associated molecular patterns. 15,16 Cooperative interactions between TLRs allow further discrimination. For example, a widely used synthetic ligand, tripalmitoyl Cys-Ser-(Lys) 4 (P3C), 17 has preference for TLR2/TLR1 heterodimers, while bacterial peptidoglycans (PGN) and macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) have preference for TLR2/ TLR6 heterodimers.TLRs interact with ligands as homodimers or, as noted for TLR2, heterodimers, and thus recruit cytosolic adaptor molecules that include myeloid differentiation protein 88 (MyD88), MyD88-adaptor like/TIR-associated protein (MAL/TIRAP), Toll-receptorassociated activator of interferon (TRIF), and Toll-receptorassociated molecule (TRAM). 16,18,19 The recruitment of MyD88 by TLR2 and TLR4, the focus of the present study, results in sequential activation of protein kinase interleukin-1 (IL-1) receptorassociated kinases (IRAKs) and transforming growth factor-␤-activated kinase (TAK1). TAK1, in turn, regulates activation of mitogen-activated protein (MAP) kinases and the transcription factor, nuclear factor B (NF-B), with ensuing production of inflammatory cytokines. TLR4 also activates a MyD88-independent pathway through TRAM and TRIF that uses interferon (IFN)-regulatory factor (IRF3) and NF-B to induce production of IFN-␤. In addition, TLRs engage signaling molecules that are commonly used by other types of receptors, such as phosphatidylinositol 3-kinase (PI3K), AKT, and possibly Src kina...

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 TGFβ-activated kinase1 which was then transmitted downstream through JNK, p38 MAP kinase, and AP1. 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 AP1 but augmented the activation of these transcription factors by Ag or thapsigargin. Robust stimulation of NFκB transcriptional activity by IL-33 was also essential for synergy. These and pharmacologic data suggested that the enhanced production of cytokines resulted in part from amplification of the activation of AP1 and NFAT as well as co-operative interactions among transcription factors. IL-33 may retune mast cell responses to Ag towards enhanced cytokine production and thus determine the symptoms and severity of Ag-dependent allergic and autoimmune diseases.

IAH provokes the release of pro-inflammatory cytokines which may serve as a second insult for the induction of MOF.

SUMMARY IL-33 is elevated in afflicted tissues of patients with mast cell-dependent chronic allergic diseases. Based on its acute effects on mouse mast cells (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. We now find that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to antigen. This reduction required >72 h exposure to IL-33 for onset and 1–2 wk for reversion following IL-33 removal. This hypo-responsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation including antigen-mediated calcium mobilization and cytoskeletal reorganization; potentially as a consequence of down-regulation of the expression of PLCγ1 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 down-regulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease.

BackgroundThe study of the endotoxin tolerance phenomenon in light of the recently defined roles of mast cells and toll-like receptors as essential components of the innate immune response and as orchestrators of acquired immunity may reveal potentially useful mechanisms of immunomodulation of infectious and allergic inflammatory responses, such as sepsis or asthma. Here we evaluated the phenomenon of direct tolerance of endotoxins, as well as the induction of cross-tolerance and synergism by stimulation with toll-like receptor-2 (TLR2) and FcεR1 agonists, in murine mast cells prestimulated with lipopolysaccharide (LPS). Additionally, we evaluated some stimulatory and inhibitory signaling molecules potentially involved in these phenomena.MethodsMC/9 cells and primary bone marrow-derived mast cells obtained from C57BL/6 and TLR4-/- knock-out mice were sensitized to DNP-HSA (antigen) by incubation with DNP-IgE and were prestimulated with LPS for 18 hr prior to stimulation. Cultures were stimulated with LPS or Pam3Cys-Ser-(Lys)4 3HCl (P3C), a TLR2 agonist, individually or in combination with antigen. The production of IL-6 and TNFα, the phosphorylation of NFκB and p38 MAPK, and the expression of TLR4 and SOCS-1 and -3 were analyzed.ResultsWe found that production of TNFα and IL-6 in murine mast cells that have been pretreated with LPS and challenged with TLR4 (LPS) or -2 (P3C) agonists was reduced, phenomena described as endotoxin tolerance (LPS) and cross-tolerance (P3C), respectively. The expression of TLR4 was not affected by LPS pretreatment. Our results show that the FcεR1 agonist DNP-HSA (antigen) interacts synergistically with LPS or P3C to markedly enhance production of cytokines (TNFα and IL-6). This synergistic effect with LPS and P3C was also attenuated by LPS pretreatment and was mediated by TLR4. These results may be attributed to the reduction in phosphorylation of the mitogen-activated protein kinase (MAPK), p38, and the transcription factor NFκB, as well as to an increase in the expression of the suppressors of cytokine signaling (SOCS)-1 and -3 proteins in LPS-pretreated mast cells.ConclusionsThese findings can be explored with respect to the modulation of inflammatory responses associated with infectious and allergic processes in future studies.

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W v or Wsh/Wsh) and the adoptive transfer of mast cells.Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-β, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.Keywords: Mast cell r Regulatory T cell r Tc17 cell r Th17 cell r Type 1 diabetes (T1D)Additional supporting information may be found in the online version of this article at the publisher's web-site Eur. J. Immunol. 2015Immunol. . 45: 2873Immunol. -2885 Introduction Type 1 diabetes (T1D) is an autoimmune disease that currently affects approximately 10-20 million people and is increasing in incidence worldwide. Similar to other autoimmune dysfunctions, the etiology of T1D is obscure. However, the development of the disease has been shown to be influenced by genetic and environmental factors including viral infections, food antigens, vaccinations, toxins, and stress [1,2]. T1D is characterized by pancreatic dysfunction resulting from the specific destruction of insulin-producing pancreatic β cells in the islets of Langerhans by autoreactive T lymphocytes. The onset of the hyperglycemia is preceded by a preclinical period designated as insulitis due to the infiltration of mononuclear cells, including T and B lymphocytes, monocytes, dendritic cells, and natural killer cells, into the pancreatic islets. Additionally, mast cell accumulation into the pancreatic islets has been reported prior to and at the onset of T1D in diabetic BB rats [3,4]. Regulatory T (Treg) cells are crucial for preventing the development of autoimmune diseases, maintaining self-tolerance and regulating the induction and intensity of the immune response to selfmolecules [5]. Previous studies have described the importance of Treg cells in the suppression of autoimmunity in an experimental T1D model, while specific IL-17-producing helper T cells (Th17) have been implicated in the promotion of the disease [6][7]...

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