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TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation.

ABSTRACT: Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-? neutralizing antibody on compound 48/80-provoked thermal hyperalgesia.We treated ND4 Swiss male mice with intravenous anti-TNF-? antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-? gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue.We found that TNF-? neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-? neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-? protein or mRNA levels were detected within 3 hours of administration of compound 48/80. The neutralizing antibodies likely target pre-formed TNF-? including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-?, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These early effects on nociceptors are abrogated by TNF-? blockade, resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli.

SUBMITTER: Chatterjea D

PROVIDER: S-EPMC3869523 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation.

Chatterjea Devavani D Paredes Luisa L Martinov Tijana T Balsells Evelyn E Allen Juliann J Sykes Akilah A Ashbaugh Alyssa A

F1000Research 20130821

<h4>Background</h4>Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperal ...[more]

PMID: 24555087

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Project description:Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.

Project description:BackgroundMast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood.ObjectiveWe investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells.MethodsHuman umbilical cord blood-derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies.ResultsStimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin.ConclusionHuman mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

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TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation.

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TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation.

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