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Front. Immunol. | doi: 10.3389/fimmu.2018.02193

An optimized protocol for the isolation and functional analysis of human lung mast cells.

 Avinash Ravindran1*,  Elin Ronnberg1,  Joakim Dahlin1, Luca Mazzurana2, 3, Jesper Safholm2, 4, Ann-charlotte Orre2, Mamdoh Al-Ameri2, Peter Peachell5,  Mikael Adner2, 4,  Sven-Erik Dahlén2, 4,  Jenny Mjösberg2, 3, 6 and  Gunnar Nilsson1, 7*
  • 1Immunology and Allergy Unit, Department of Medicine Solna, Karolinska University Hospital, Sweden
  • 2Thoracic Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet (KI), Sweden
  • 3Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet (KI), Sweden
  • 4Unit for Experimental Asthma and Allergy Research, Centre for Allergy Research, The Institute of Environmental Medicine,, Karolinska Institutet (KI), Sweden
  • 5Academic Unit of Respiratory Medicine, The Royal Hallamshire Hospital, Sheffield, UK, University of Sheffield, United Kingdom
  • 6Department of Clinical and Experimental Medicine, Linköping University, Sweden
  • 7Department of Medical Sciences, Uppsala University, Sweden

Background:
Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, FcεRI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield.

Aim:
To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression.

Methods:
Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry.

Results:
We observed a significant increase in the yield of total human lung CD45+ immune cells and an even more pronounced increase in the yield of CD117+ mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods.

Conclusion:
The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Keywords: Mast Cells, Lung, enzymatic digestion protocols, human mast cells, Mast cell isolation

Received: 06 Jun 2018; Accepted: 05 Sep 2018.

Edited by:

Jagadeesh BAYRY, Institut National de la Santé et de la Recherche Médicale (INSERM), France

Reviewed by:

Axel Lorentz, University of Hohenheim, Germany
Nicolas Gaudenzio, Institut National de la Santé et de la Recherche Médicale (INSERM), France  

Copyright: © 2018 Ravindran, Ronnberg, Dahlin, Mazzurana, Safholm, Orre, Al-Ameri, Peachell, Adner, Dahlén, Mjösberg and Nilsson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Mr. Avinash Ravindran, Karolinska University Hospital, Immunology and Allergy Unit, Department of Medicine Solna, Stockholm, Sweden, avinash.ravindran@ki.se
Prof. Gunnar Nilsson, Karolinska University Hospital, Immunology and Allergy Unit, Department of Medicine Solna, Stockholm, Sweden, Gunnar.P.Nilsson@ki.se