Sarah K. Norton

Fullerene Nanomaterials Inhibit the Allergic Response

Fullerenes are a class of novel carbon allotropes that may have practical applications in biotechnology and medicine. Human mast cells (MC) and peripheral blood basophils are critical cells involved in the initiation and propagation of several inflammatory conditions, mainly type I hypersensitivity. We report an unanticipated role of fullerenes as a negative regulator of allergic mediator release that suppresses Ag-driven type I hypersensitivity. Human MC and peripheral blood basophils exhibited a significant inhibition of IgE dependent mediator release when preincubated with C60 fullerenes. Protein microarray demonstrated that inhibition of mediator release involves profound reductions in the activation of signaling molecules involved in mediator release and oxidative stress. Follow-up studies demonstrated that the tyrosine phosphorylation of Syk was dramatically inhibited in Ag-challenged cells first incubated with fullerenes. In addition, fullerene preincubation significantly inhibited IgE-induced elevation in cytoplasmic reactive oxygen species levels. Furthermore, fullerenes prevented the in vivo release of histamine and drop in core body temperature in vivo using a MC-dependent model of anaphylaxis. These findings identify a new biological function for fullerenes and may represent a novel way to control MC-dependent diseases including asthma, inflammatory arthritis, heart disease, and multiple sclerosis.

Cutting Edge: CD4 T Cell-Mast Cell Interactions Alter IgE Receptor Expression and Signaling

Mast cell activation is associated with atopic and inflammatory diseases, but the natural controls of mast cell homeostasis are poorly understood. We hypothesized that CD4+CD25+ regulatory T cells (Treg) could function in mast cell homeostasis. In this study, we demonstrate that mast cells can recruit both Treg and conventional CD4+ T cells (Tconv). Furthermore, Treg, but not Tconv, suppress mast cell FcεRI expression. Despite the known inhibitory functions of IL-10 and TGFβ1, FcεRI suppression was independent of IL-10 and TGF-β1 and required cell contact. Surprisingly, coculture with either Treg or Tconv cells suppressed IgE-mediated leukotriene C4 production but enhanced cytokine production by mast cells. This was accompanied by a selective increase in FcεRI-mediated Stat5 phosphorylation, which is a critical mediator of IgE-mediated cytokine secretion. These data are the first direct demonstration that mast cells can recruit Treg and illustrate that T cell interactions can alter the mast cell response.

IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcεRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcεRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcεRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.

A potential new target for asthma therapy: a disintegrin and metalloprotease 10 (ADAM10) involvement in murine experimental asthma.

To cite this article: Mathews JA, Ford J, Norton S, Kang D, Dellinger A, Gibb DR, Ford AQ, Massay H, Kepley CL, Scherle P, Keegan AD, Conrad DH. A potential new target for asthma therapy: A Disintegrin and Metalloprotease 10 (ADAM10) involvement in murine experimental asthma. Allergy 2011; 66: 1193–1200.

Uptake and distribution of fullerenes in human mast cells

Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C(70)) that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and the duration they can be detected in situ. We show that C(70)-TR are nonspecifically endocytosed into MCs, where they are shuttled throughout the cytoplasm, lysosomes, mitochondria, and into endoplasmic reticulum at different times. No nuclear or secretory granule localization was observed. The C(70)-TR remained detectable within cells at 1 week. These studies show that MCs endocytose fullerenes, where they are shuttled to organelles involved with calcium and reactive oxygen species production, which may explain their efficacy as cellular inhibitors. From the clinical editor: Fullerenes are carbon cages of variable size that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation. These were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and duration, demonstrating that MCs endocytose fullerenes, which are shuttled to organelles involved with calcium and reactive oxygen species production. This intracellular trafficking may explain the efficacy of fullerenes as cellular inhibitors.

IL-4 and TGF-β1 Counterbalance One Another while Regulating Mast Cell Homeostasis

Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-β1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

Myeloid-derived suppressor cells enhance IgE-mediated mast cell responses

Mast cells and MDSCs are increased by parasitic infection and tumor growth. We previously demonstrated that enhanced MDSC development in ADAM10 transgenic mice yielded resistance to Nb infection and that coculturing MDSCs and mast cells enhanced cytokine production. In the current work, we show that MDSC‐mast cell coculture selectively enhances IgE‐mediated cytokine secretion among mast cells, without increasing MDSC cytokine production. This effect was independent of cell contact and elicited by Ly6C+ and Ly6C/G+ MDSC subsets. These interactions were functionally important. MDSC depletion with the FDA‐approved drug gemcitabine exacerbated Nb or Trichinella spiralis infection and reduced mast cell‐dependent AHR and lung inflammation. Adoptive transfer of MDSC worsened AHR in WT but not mast cell‐deficient Wsh/Wsh mice. These data support the hypothesis that MDSCs enhance mast cell inflammatory responses and demonstrate that this interaction can be altered by an existing chemotherapeutic.