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Exocytic machineries differentially control mediator release from allergen-triggered RBL-2H3 cells

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Abstract

Background

Mast cells utilize SNAREs (soluble-N-ethyl-maleimide sensitive factor attachment protein receptors) and SM (Sec1/Munc18) proteins to secrete/exocytose a variety of proinflammatory mediators. However, whether a common SNARE-SM machinery is responsible remains unclear.

Methods

Four vesicle/granule-anchored SNAREs (VAMP2, VAMP3, VAMP7, and VAMP8) and two Munc18 homologs (Munc18a and Munc18b) were systematically knocked down or knocked out in RBL-2H3 mast cells and antigen-induced release of β-hexosaminidase, histamine, serotonin, and TNF was examined. Phenotypes were validated by rescue experiments. Immunofluorescence studies were performed to determine the subcellular distribution of key players.

Results

The reduction of VAMP8 expression inhibited the exocytosis of β-hexosaminidase, histamine, and serotonin but not TNF. Unexpectedly, however, confocal microscopy revealed substantial co-localization between VAMP8 and TNF, and between TNF and serotonin. Meanwhile, the depletion of other VAMPs, including knockout of VAMP3, had no impact on the release of any of the mediators examined. On the other hand, TNF exocytosis was diminished specifically in stable Munc18bknockdown cells, in a fashion that was rescued by exogenous, RNAi-resistant Munc18b. In line with this, TNF was co-localized with Munc18b (47%) to a much greater extent than with Munc18a (13%).

Conclusion

Distinct exocytic pathways exist in mast cells for the release of different mediators.

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Data availability

All data generated or analysed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

This work was supported by the National Institute of Allergy and Infectious Diseases Grant R15AI133430-01 to H.X. and S.S, and by the Mississippi INBRE, which was funded by an Institutional Development Award from the National Institute of General Medical Sciences under grant no. P20GM103476. We thank Dr. Seth Malmersjö for kindly providing the RNAi-resistant VAMP8 rescue construct and Dr. Jonathan Lindner at the INBRE Imaging Facility for excellent technical support.

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Author notes

  1. Pratikshya Adhikari and Tolulope E. Ayo have contributed equally to the manuscript.

Authors and Affiliations

  1. Center for Molecular and Cellular Biosciences, School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi, Hattiesburg, MS, 39406, USA

    Pratikshya Adhikari, Tolulope E. Ayo, John C. Vines & Hao Xu

  2. Division of Fundamental Neurobiology, University Health Network, Toronto, ON, M5T 2S8, Canada

    Shuzo Sugita

  3. Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, ON, M5S 1A8, Canada

    Shuzo Sugita

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Contributions

All authors contributed to the study conception and design. Material preparation was performed by PA, TA, and SS. Data collection and analysis were conducted by PA, TA, HX, and JV. The first draft of the manuscript was written by HX, PA, and TA. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Hao Xu.

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Adhikari, P., Ayo, T.E., Vines, J.C. et al. Exocytic machineries differentially control mediator release from allergen-triggered RBL-2H3 cells. Inflamm. Res. 72, 639–649 (2023). https://doi.org/10.1007/s00011-023-01698-z

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  • DOI: https://doi.org/10.1007/s00011-023-01698-z

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