Elsevier

Molecular Immunology

Volume 46, Issue 13, August 2009, Pages 2505-2514
Molecular Immunology

Ragweed pollen-mediated IgE-independent release of biogenic amines from mast cells via induction of mitochondrial dysfunction

https://doi.org/10.1016/j.molimm.2009.05.023Get rights and content

Abstract

Normal functions of mitochondria are required for physiological dynamics of cells, while their dysfunction contributes to development of various disorders including those of immune system. Here we demonstrate that exposure of mast cells to ragweed pollen extract increases production of H2O2 via mitochondrial respiratory complex III. These mitochondrial ROS (mtROS) enhance secretion of histamine and serotonin from mast cells, but not enzymes such as β-hexosaminidase, independently from FcɛRI-generated stimuli. The release of biogenic amines is associated with inhibition of secretory granules’ H+-ATPase activity, activation of PKC-δ and microtubule-dependent motility, and it is independent from intracellular free Ca2+ levels. To asses differences from IgE-mediated mast cell degranulation we show that mtROS decrease antigen-triggered β-hexosaminidase release, while they are synergistic with antigen-induced IL-4 production in sensitized cells. Taken together, these data indicate that mitochondrial dysfunction can act independently from adaptive immunity, as well as augments Th2-type responses. Pharmacological maintenance of physiological mitochondrial function could have clinical benefits in prevention and treatment of allergic diseases.

Introduction

Mast cells are one of the most important effectors involved in elicitation of allergic inflammation and immune responses to many pathogens including parasites (Metcalfe et al., 1997). Antigenic activation of mast cells via the high-affinity receptor for IgE (FcɛRI) mediates exocytosis of cytoplasmic granules containing preformed mediators, secretion of lipid-derived factors, and de novo synthesis of cytokines, chemokines and growth factors (Burgoyne and Morgan, 2003, Logan et al., 2003, Metcalfe et al., 1997, Rivera and Gilfillan, 2006). In addition to FcɛRI-mediated signals, exposure to a variety of stimuli can lead to the release of mast cell mediators (Frossi et al., 2004). Pathogen-associated molecules may activate mast cells and basophils via receptors selectively expressed on their surfaces (Kojima et al., 2007). Eosinophil-derived major basic protein, compound 48/80 or substance P also induces degranulation of mast cells (Munitz et al., 2003). Several lines of evidence indicate that oxidative stress is also a stimulus for mast cell activation (Frossi et al., 2003, Ohmori et al., 1979, Swindle et al., 2002). During allergic and other inflammatory reactions mast cells are exposed to an oxidative microenvironment because ROS are produced by various cell types in the peripheral tissues as a consequence of their effector function (Nagata, 2005). We have previously reported that pollen grains, sub-pollen particles, and pollen extracts contain intrinsic NAD(P)H oxidases, which generate ROS [superoxide anions (O2radical dot)] (Bacsi et al., 2006a, Boldogh et al., 2005). These radicals induce oxidative stress in cultured cells, as well as in airway and conjunctival epithelium within minutes of exposure (Bacsi et al., 2005, Boldogh et al., 2005).

There is a close correlation between the exclusively maternal inheritance of mitochondria and the fact that maternal history of atopy and asthma is one of the substantial risk factors for the development of asthma in children (Litonjua et al., 1998). A mitochondrial haplogroup has been shown to be associated with elevated total serum IgE levels in asthmatic patients (Raby et al., 2007). Oxidative stress and mitochondrial metabolism are involved in antigen-induced release of mast cell mediators, including IL-4, which is essential for naive T cell polarization toward Th2 phenotype (Frossi et al., 2003, Inoue et al., 2008). Studies with metabolic inhibitors have demonstrated a close link between mitochondrial energy production and mast cell degranulation (Johansen, 1987). Furthermore, release of Ca2+ from mitochondria is involved in antigen-induced mast cell degranulation (Suzuki et al., 2006).

Here we report for the first time that treatment with short ragweed (Ambrosia artemisiifolia) pollen extract (RWE) induces elevated mitochondrial ROS (mtROS) production in non-sensitized RBL-2H3 cells, a model of mucosal mast cells (Park and Beaven, 2009, Seldin et al., 1985). We show that increased production of ROS from mitochondrial respiratory complex III, but not intrinsic pollen NAD(P)H oxidase-generated ROS directly, enhances secretion of histamine and serotonin from non-sensitized mast cells. Mitochondrial ROS trigger the release of biogenic amines, but not enzymes such as β-hexosaminidase, via inducing PKC-δ- and microtubule-dependent motility of secretory granules and inhibiting activity of vacuolar H+-ATPase independently from intracellular Ca2+ levels. We demonstrate that mtROS are also able to enhance FcɛRI-mediated IL-4 production of mast cells. These findings may shed light on a new role for mitochondrial dysfunction in the regulation of mast cell activation.

Section snippets

Reagents

All reagents were purchased from Sigma–Aldrich (St. Louis, MO) unless otherwise stated.

Cell cultures

The RBL-2H3 cells were obtained from the American Type Cell Collection and cultured at 37 °C in a humidified atmosphere with 5% CO2 in Minimum Essential Medium containing Earle's salts and l-glutamine (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml).

Analysis of inflammatory mediator release

Cells were treated with RWE (Greer Laboratories, Lenoir, NC) at the indicated concentrations

RWE induces the release of biogenic amines in an IgE-independent manner

Previous studies have demonstrated that exposure to a variety of non-antigenic stimuli leads to the activation of mast cells (Frossi et al., 2004, Stassen et al., 2002, Yoshimaru et al., 2006). Here we investigated whether RWE induces degranulation of RBL-2H3 cells in the absence of sensitization with IgE antibodies. Treatment of the cells with RWE induced the release of histamine in a dose-dependent manner (Fig. 1A). Administration of RWE (100 μg/ml) to the cells also increased the secretion of

Discussion

Mitochondria are involved in regulation of various cellular functions as they store/release/generate signaling mediators including Ca2+ and ROS. These mediators are required in differentiation and function of various immune cells participating in innate and adaptive responses (Del Prete et al., 2008, Hunt et al., 1991). In this study, we show that increased production of mtROS, induced by treatment with RWE, triggers secretion of biogenic amines, i.e. histamine and serotonin, but not

Acknowledgements

This work was supported by NIAID, P01 AI062885-01 (I.B., S.S., T.H.), NIH HL071163 (S.S., I.B.), NIEHS Center Grant, EOS 006677 and the Hungarian Scientific Research Fund (73347).

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