Basic and clinical immunology
Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3

https://doi.org/10.1016/j.jaci.2004.03.049Get rights and content

Abstract

Background

Although mast cells (MCs) have been clearly implicated in innate immune responses involving bacteria, their ability to respond to viral infection is less clear.

Objective

Given that MCs increase at sites of inflammation and are located at surfaces where exposure to invading viruses may occur, we explored the ability of MCs to produce cytokines including type I IFNs after exposure to viruses and to polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA, and characterized the receptors involved, if any.

Methods

Human peripheral blood-derived cultured MCs and 2 MC lines, Laboratory of Allergic Disease MC line and human MC line 1, were stimulated with viruses and polyI:C, and cytokine production, degranulation, and signaling pathway activation were examined. Because polyI:C is a ligand for Toll-like receptor (TLR)–3, human MCs were also analyzed for TLR expression.

Results

Viruses and polyI:C induced IFN-α and IFN-β production. PolyI:C did not induce TNF, IL-1β, IL-5, or GM-CSF production, in contrast with other TLR ligands (LPS, peptidoglycan, CpG-A, or flagellin). IFN-α production involved nuclear factor–κB, p38, and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. RT-PCR and Western blot analysis confirmed expression of TLR-3 by all MCs. Human cultured MCs also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7 and TLR-9. Antibodies to TLR-3 significantly decreased IFN-α production. Bone marrow–derived MCs from TLR-3 knockout mice showed an ablated response to polyI:C.

Conclusions

Murine and human MCs produce type I IFNs after exposure to double-stranded RNA and/or virus, the former via specific interactions with TLR-3. These data suggest that MCs contribute to innate immune responses to viral infection via the production of type I IFNs.

Section snippets

Human MC culture

Human peripheral blood CD34+ progenitor cells were cultured as described.4 At 6 to 8 weeks of culture, aliquots of 2 × 104 cultured cells were spun onto glass slides (Cytospin 2; Thermo Electron Corporation, Houston, Tex) and stained with toluidine blue.4 More than 99% of the nonadherent cells contained metachromatic granules, and flow cytometry showed them to be positive for Kit and FcεRI receptors.

Human MC line 1 (HMC-1) cells were cultured in Iscove medium containing 10% FBS, 100 U/mL

dsRNA induces IFN-α production by human MCs

Double-stranded RNA, a synthetic mimic of viral RNA, has been shown to induce type I IFNs in several cell types, including dendritic cells.8., 9. For these studies, we stimulated LAD, HMC-1, and HCMCs with polyI:C for 0 to 24 hours, and total RNA samples were analyzed for the presence of IFN-α and IFN-β mRNA by using RT-PCR. PolyI:C induced IFN-α and IFN-β mRNA expression in LAD and HMC-1 cells, with gene induction occurring as early as 30 minutes (Fig 1, A). HCMCs constitutively expressed low

Discussion

This is the first report that human MCs express TLR-3 and are capable of responding to the TLR-3 ligand, polyI:C dsRNA, by producing IFN-α, a molecule heretofore not known to be synthesized and released by MCs of any species. This observation may be important for understanding innate host responses to viral infections in peripheral tissues. IFN-α effects are mediated by both direct inhibition of viral replication and activation of nearby immune cells such as NK cells, macrophages, and T cells,

Acknowledgements

We thank Brian Kelsall for reovirus and influenza virus and for critically revising this article. We also thank Helene Rosenberg for RSV and technical advice. Dr Flavell is an investigator of the Howard Hughes Medical Institute.

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    Supported by the intramural program at the National Institutes for Health Research. Dr Alexopoulou was supported in part by a National Institutes of Health grant (P01AI36529).

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