An unexpected role for autophagy in degranulation of mast cells

Autophagic Punctum

Autophagic Punctum

Autophagy 7:6, 657-659; June 2011; © 2011 Landes Bioscience

An unexpected role for autophagy in degranulation of mast cells Hiroyasu Nakano1 and Hiroko Ushio2 Department of Immunology and 2Atopy (Allergy) Research Center; and Juntendo University School of Medicine; Bunkyo-ku, Tokyo Japan

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ast cells play a crucial role in allergic inflammatory reactions through releasing cytosolic granules upon antigen stimulation. However, the mechanisms underlying maturation and release of secretory granules are not fully understood. We found that autophagy is constitutively induced in mast cells under full nutrition conditions, and type II LC3 (LC3-II), a marker for autophagosomes, localizes on secretory granules. While deletion of Atg7 does not impair the development of bone marrow-derived mast cells (BMMCs), Atg7-deficient BMMCs show severe impairment of degranulation, but not cytokine production, upon antigen stimulation. Moreover we found that LC3-II, but not LC3-I, colocalizes with CD63, a marker for secretory lysosomes and is released extracellularly along with degranulation in wild-type BMMCs, but not Atg7-deficient BMMCs. Finally, passive cutaneous anaphylaxis reactions are almost completely abolished in mast celldeficient mice reconstituted with Atg7deficient BMMCs. Collectively, these results suggest that autophagy is not essential for the development, but plays a crucial role in degranulation, of mast cells.

granules that are induced by antigen stimulation. Secretory granules in mast cells are considered to be secretory lysosomes that bridge the secretory and endocytic pathways. However, the contribution of autophagy to granule maturation and degranulation of mast cells is totally unknown. We found that the conversion of LC3-I to LC3-II is constitutively induced in both freshly prepared and bone marrow-derived mast cells under full nutrient conditions, and LC3-II localizes in secretory granules of mast cells. Transmission electron microscopy analysis of BMMCs revealed that there are a large number of granules filled with electron-dense materials and/ or small vesicles, but not typical autophagosomes under full nutrient conditions, suggesting that LC3-II localizes in the granules of mast cells. We next tested whether autophagy may play a role in the development of mast cells. Considering that Atg7-/- mice die soon after birth, we generated Atg7-/- BMMCs prepared from interferon-inducible Atg7-deficient mice, in which expression of the Atg7 gene is deleted in BM cells upon poly I:C injection. Whereas conversion of LC3-I to LC3-II is completely blocked in Atg7-/BMMCs, differentiation and proliferation of Atg7-/- BMMCs appears to be normal. Consistent with a previous study using hepatocytes, LC3-I forms large aggregates in Atg7-/- BMMCs, whereas LC3-II shows a punctate distribution in wildtype BMMCs. Since LC3-II appears to localize in granules, we next examined whether degranulation is impaired in Atg7-/- BMMCs. Interestingly, FcεRIinduced release of both β-hexosaminidase and histamine are severely impaired in Atg7-/- BMMCs. In sharp contrast,

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Key words: Atg7, mast cells, light chain 3, CD63, degranulation, histamine Submitted: 03/01/11 Revised: 03/04/11 Accepted: 03/07/11 DOI: 10.4161/auto.7.6.15384 Correspondence to: Hiroyasu Nakano and Hiroko Ushio; Email: [email protected] and [email protected] Punctum to: Ushio H, Ueno T, Kojima Y, Komatsu M, Tanaka S, Yamamoto A, et al. Crucial role for autophagy in degranulation of mast cells. J Allergy Clin Immunol 2011; 127:1267–1276.e6; PMID: 21333342; DOI: 10.1016/j.jaci.2010.12.1078.

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Mast cells express high-affinity receptors for IgE (FcεRI) on the cell surface and release histamine and other preformed chemical mediators upon cross-linking of FcεRI receptors by polyvalent antigen, thereby playing a crucial role in allergic inflammatory reactions. Mast cells are also involved in protection of the host from bacterial infection. Most of these functions of mast cells are mediated by regulated exocytosis of specific secretory

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Figure 1. A model of a role for autophagy in maturation and/or release of secretory granules in mast cells. Autophagy is constitutively induced in mast cells. Some portions of LC3-II- and CD63-positive granules may be initially generated from small vesicles that sequester a set of contents of secretory granules such as histamine and β-hexosaminidase. These small vesicles may be eventually engulfed by MVBs, resulting in mature secretory granules. Upon antigen stimulation, these secretory granules containing LC3-II- and CD63-positive small vesicles are released extracellularly.

FcεRI-induced phosphorylation of early signaling molecules, Ca 2+ mobilization or production of proinflammatory cytokines are not impaired. These results suggest that deficiency of autophagy results in a relatively specific defect in degranulation, but not global dysfunction of mast cells. Taking into account that FcεRIinduced degranulation is selectively impaired in Atg7-/- BMMCs, we surmised that autophagy may play a role in granule maturation, translocation, and/or fusion of granules with the plasma membrane along with degranulation. A previous study has shown that a member of the tetraspanin family, CD63 (also known as LAMP-3) localizes on granule membrane of basophils, mast cells and platelets, and is considered to be a secretory lysosomal marker. Moreover, expression of CD63 on the cell surface is upregulated along with degranulation through granuleplasma membrane fusion. Thus, we investigated whether LC3-II colocalizes with CD63. Intriguingly, LC3-II colocalizes with CD63 in wild-type BMMC, but not in Atg7-/- BMMCs, in which LC3-I forms large aggregates or diffusely distributes in the cytoplasm. We then tested

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whether FcεRI-induced upregulation of CD63 on the cell surface is impaired in Atg7-/- BMMCs. Whereas surface expression of CD63 on wild-type BMMCs is almost equivalent to Atg7-/- BMMCs before stimulation, upregulation of CD63 upon stimulation is decreased in Atg7-/BMMCs compared to wild-type BMMCs. These results suggest that granule translocation or fusion of granules with the plasma membrane is partially impaired in Atg7-/- BMMCs. LC3-II accumulates in granules and colocalizes with CD63, prompting us to test whether LC3-II may be released into the extracellular space like exosomes along with degranulation. Using BMMCs stably expressing LC3 tagged with the red fluorescent protein Cherry (Cherry-LC3), we found that Cherry-LC3 is released extracellularly upon stimulation in wildtype BMMC. However, such release is not detected in Atg7-/- BMMC. Moreover, large amounts of CD63 are detected in the released granule fractions; however, such release of CD63 is significantly reduced in Atg7-/- BMMCs. These results suggest that LC3-II- and CD63-positive granules are released extracellularly like

Autophagy

exosomes. One plausible interpretation would be that at least some portion of LC3-II- and CD63-positive granules may be initially generated from small vesicles that sequester a set of contents of secretory granules such as histamine and β-hexosaminidase (Fig. 1). These small vesicles may be eventually engulfed by multivesicular bodies (MVBs), resulting in mature secretory granules. Consistently, recent studies have shown that selective autophagy collects cargos that are subsequently engulfed by multivesicular endosomes, which eventually fuse with the plasma membrane to release their contents extracellularly. We finally adoptively transferred wild-type and Atg7-/- BMMCs into skins of mast cell-deficient mice. We assessed degranulation of reconstituted mast cells by passive cutaneous anaphylaxis (PCA) reactions to evaluate degranulation of mast cells in vivo. PCA reaction is almost completely abolished in mast cell-deficient mice reconstituted with Atg7-/- BMMCs. Together, our findings underscore an important role for autophagy in maturation and/or release of secretory granules in mast cells. Mast cells play crucial roles

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in the protection of hosts from bacterial infection as well as various allergic diseases, suggesting that a strategy to inhibit global function of mast cells might cause

serious side effects through exacerbating bacterial infection. Given that inhibition of autophagy might selectively impair antigen-induced degranulation, but not

cytokine production, autophagy might be a potential target to treat allergic diseases through selective suppression of antigeninduced degranulation of mast cells.


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