BASOPHILS AND MAST CELLS : methods and protocols.

378 Pages·2020·10.2 MB·English

Methods in
Molecular Biology 2163
Bernhard F. Gibbs
Franco H. Falcone Editors
Basophils and
Mast Cells
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas
thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall
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step fashion, opening with an introductory overview, a list of the materials and reagents
neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
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series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexedinPubMed.
Basophils and Mast Cells
Methods and Protocols
Second Edition
Edited by
Bernhard F. Gibbs
Division of Experimental Allergology and Immunodermatology, Department of Human Medicine,
School of Medicine and Health Sciences, Carl von Ossietzky University of Oldenburg, Oldenburg, Germany
Franco H. Falcone
Institute for Parasitology, Justus Liebig University Giessen, Biomedizinisches Forschungszentrum
Seltersberg (BFS), Giessen, Germany
Editors
BernhardF.Gibbs FrancoH.Falcone
DivisionofExperimentalAllergology InstituteforParasitology
andImmunodermatology JustusLiebigUniversityGiessen,Biomedizinisches
DepartmentofHumanMedicine ForschungszentrumSeltersberg(BFS)
SchoolofMedicineandHealthSciences Giessen,Germany
CarlvonOssietzky
UniversityofOldenburg
Oldenburg,Germany
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0695-7 ISBN978-1-0716-0696-4 (eBook)
https://doi.org/10.1007/978-1-0716-0696-4
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Acknowledgements
The cover image shows a peripheral blood basophil immunostained with BB1 antibody,
showingthepresenceofbasogranulin(green)inthesecretorygranules(copyrightAndrewF
WallsandAkinoriMochizuki).
v
Preface to the Second Edition
Thewell-receivedfirsteditionofourbook,BasophilsandMastCells:MethodsandProtocols,
highlights the need for detailed methods that focus on basophils and mast cells, especially
the use of primary cells. This second edition builds on and extends the strengths of the
previous one. The large majority of chapters have been revisited and, where necessary,
updatedbytheiroriginalauthors.Inadditiontorefiningexistingmethods,thenewedition
alsocontainsnewlydevelopedmethodsprovidedbycutting-edgeresearchersinthefield.
To accommodate the new chapters, we have expanded the structure of the book from
originallyfourintosixparts:afteranintroductiontothebasicbiologyofbasophilsandmast
cells in Part I, Part II focuses on obtaining the cells by purification or culture of stem cells
progenitors.Here,anewchapterhasbeenaddedbySiebenhaarandcolleagues,describing
the generation of peripheral CD34+ stem cell-derived mast cells (Chapter 5). Similarly,
MacGlashandescribestwoprotocolsfor thegenerationofbasophilsfromCD34+progeni-
tors,startingwithtwodifferentmaterials:theconvenient(butmoreexpensive)commercial
sources of CD34+ progenitors, or leukapheresis pack. As an interesting aside, MacGlashan
remindsusinhisintroductiontoChapter6thatalthoughbasophilsarenormallyconsidered
short-lived cells, their residence time in circulation (with estimates ranging from 12 h to a
fewdays)ismorelikelytobejustthetipoftheiceberg,asamuchlongertimeisspentduring
the process leading to maturation in the bone marrow, before they are released into
circulation.Our understandinghereisverypatchy.
PartIIIofthebookisnowdedicatedtobasophilandmastcelllines.After theupdated
summaryofthemostcommonlyusedmastcellandbasophillinesbyPassante(Chapter10),
threenewchaptersdescribetheuseofhumanizedreportersystems.Asapossiblealternative
to BATs and MATs, described in the next part, which require speedy processing of blood
samplesandflowcytometryequipment,IgEreportersystemsarealsoavailable.Thesecanbe
combinedwithallergenarrays,asdescribedbyKalliandcoauthorsinChapter12.Barwary
et al. (Chapter 13) describe a new, easy to measure humanized IgE reporter system. Here,
thecellsarepreloadedwitharedfluorescentproteintargetedtothegranules,whichisthen
releaseduponactivation,andcanbeconvenientlymeasuredwithouttheneedforexpensive
reagentsorlongincubationtimes.Ofcourse,otherresearcharenashavealsoadvancedsince
thepublicationofourfirstedition,suchasbiomedicalnanotechnology,andwethereforefelt
thatitwasimportanttoincludeprotocolstailoredtothestudyoftheinteractionsbetween
nano-orsubmicron-scalematerialsandbasophilsormastcells,asinChapter14byAlelwani
andhercoauthors.
PartIVdescribesdiagnosticapplications.Thisparthasalsobeenexpandedandupdated.
The new Chapter 16 by Ebo and coauthors now introduces BATs in more depth, in
particular with respect to their use in understanding drug hypersensitivity reactions. Two
usefulflowcytometrytechniquesappliedtobasophilsarepresentedbyElstandcoauthors:
in Chapter 17, a technique which, rather than detecting changes in surface expression of
activation markers such as CD63 or CD203c, allows staining of exteriorized granular
materials. The process takes advantage of the ability of fluorescently labeled avidin to bind
tonegativelychargedproteoglycanscontainedinthegranulematrix,whichareexteriorized
during degranulation; the method correlates well with CD63 upregulation [1]. In
Chapter 18, the same authors remind us that not all activation pathways for mast cells and
vii
viii PrefacetotheSecondEdition
basophils are mediated via crosslinking of the high affinity IgE receptor and describe a
protocol for the assessment of Mas-related G protein-coupled receptor (MRGPRX2)
expression on the two cell types. The MRGPRX2 receptor is increasingly recognized as
being involved in many systemic pseudo-allergic (or anaphylactoid) reactions to small
molecules, such as basic secretagogues and several drugs [2]. Measuring cellular activation
byflowcytometryisbynomeanslimitedtobasophils.Similartechniquescanbealsoapplied
to mast cells: the new Chapter 19 by Bahri et al. describes a novel method for mast cell
activationtests(MATs),extendingtheinstrumentariumavailabletoresearchersinourfield.
PartVfocusesonfunctionalstudies.ThenewChapter20byBechmannetal.shouldbe
of great interest to the reader planning to study gene expression patterns in basophils; as
showninthischapter,themethodchosentopurifyRNAhasastrongbearingontheresults
(i.e.,thequalityandintegrityofRNA)obtained—animportantlessonintimeswheremany
labsrelyon“convenient”commercialkitsfor thiskindofwork.
A new chapter now enables to assess the role of basophils in lung disease. Although
basophilshavebeenknownforseveraldecadestoplayanimportantroleinasthma,andwere
describedinsputumbyKimuraandcoworkersin1975[3],studyingtheir roleindifferent
forms of asthmatic disease has been difficult due to the lack of appropriate protocols. The
techniquedescribedbyFuxetal.inChapter23willhelplaboratoriesinterestedinexamin-
ingtherolesofbasophilsindifferentasthma phenotypes.PartVIcloseswithprotocolsfor
knockoutanddiseasemodels.
In total, 11 new chapters have been added in this edition. We are confident that the
secondeditionofourbookwillprovideyouwithevenmoreessentialcutting-edgetoolsfor
yourresearchintothefunctionsofmastcellsandbasophils.Giventhatthesecellsappearto
beinvolvedinnumerousdiseasesaswellasregulatingmanyphysiologicalfunctions,thereis
a considerable unmet need to shed more light on these cell types. We hope that these
protocolswillbeofgreatbenefitandwishyoueverysuccessinyour research.
Oldenburg,Germany BernhardF.Gibbs
Giessen,Germany FrancoH.Falcone
References
1. Joulia R, Mailhol C, Valitutti S, et al (2017) Direct monitoring of basophil degranulation by using
avidin-basedprobes.JAllergyClinImmunol140:1159–1162.e6
2. McNeilBD,PundirP,MeekerS,etal(2015)Identificationofamast-cell-specificreceptorcrucialfor
pseudo-allergicdrugreactions.Nature519:237–241
3. Kimura I, Tanizaki Y, Saito K, et al (1975) Appearance of basophils in the sputum of patients with
bronchialasthma.ClinAllergy5:95–8
Preface to the First Edition
Accordingtothelatestepidemiologicalfindings,itappearsthatallergicdiseaseswillsoonbe
affectingover40%ofthepopulationinbothEuropeandelsewhere.Thereis,therefore,an
overwhelming unmet need to unravel the immunological causes of allergic inflammation
andtoimproveanti-allergictherapies.Inrecentyears,ithascometolightthatmastcellsand
theirblood-borne basophilcounterpartsnotonlycontributesignificantlytothesymptoms
ofallergybutalsoplayaroleinsupportingtheunderlyingtendencyoftheimmunesystem
torespondin apro-allergicmanner.Thisimmunomodulatoryfunctionhasparticularly led
toaresurgenceintobasophilresearchandmayonedayfinallyuncoverthebiologicalrolefor
this enigmatic and rare cell type which so readily releases a number of major inflammatory
and immunomodulatory chemical mediators within very short periods of time. Moreover,
the discovery that both mast cells and basophils potentially contribute to a variety of
diseases, such as urticaria, cancer, and autoimmune diseases, as well as physiological func-
tions,hasincreasedtheneedforimprovedtoolsfor researchingthesecells(seeChapter1).
One of the most problematic features of mast cells and basophils is their striking
differences in behavior and response to various stimulating agents and drugs depending
on their location and the species from which they are studied. This limits, to some extent,
our ability to extrapolate findings from, for example, mouse to human, but also tissue to
tissueandtheuseoftumormastcellandbasophillines.Itisthereforeimperativethatbetter
tools for the isolation of these cells from primary tissues are forthcoming. This indeed has
beenthecaseinrecentyearsandwenowpresentabookwhich,wefeel,willsignificantlyhelp
anyoneconductingresearchintothesecellsfromthemostpracticalpointofview:definitive
lab-basedprotocolstargetedtothosewhoactuallydothework!
Whether you are planning to purify basophils or mast cells from peripheral blood
(Chapter 2) or tissues (Chapter 3), or want to culture them from precursors from buffy
coatblood(Chapter4)orbonemarrow(Chapter5),youwillfindwell-testedprotocolsin
this book. Other chapters discuss the use of mast cells in organotypic skin models
(Chapter 6), the difficulty of counting basophils (Chapter 7), how to stain mast cells and
basophils in tissues (Chapter 8), how to assess mediator release (Chapter 10), and signal
transduction(Chapter15).Alsodiscussedisthesuitabilityofusingmastcellorbasophil-like
cell lines as surrogates for primary mast cells and basophils (Chapter 9) and the use of
reportercelllines(Chapter13)for thedetectionofallergen-specificIgEinserumsamples.
Chapter 12 presents protocols which can be used to measure mast cell migration using
microscopy.Chapter14describesprotocolsthatcanbeusedforgenesilencingofmastcells
andbasophils,twocelltypesthatarenotoriouslydifficulttotransfect.
Researchersmoreinterestedinexvivoorinvivoanalysiswillfindprotocolsforhuman
basophilactivationtests(BATs)inChapter11,for mastcellphenotyping(Chapter16),or
the study of murine basophils by flow cytometry in Chapter 17. Chapter 18 closes with
invivomodelsthatcanbeusedfor theanalysisofmastcellfunctions.
Our book is written by leading research scientists in the field of basophil and mast cell
biology. The impetus for this consortium of authors was given by the recently established
EU/ESF-BMBSCOSTActionBM1007“MastCellsandBasophils—TargetsforInnovative
Therapies” which we would like to acknowledge for providing the ideal platform for
scholarlycollaborationinthisfield.
ix
x PrefacetotheFirstEdition
We sincerely hope that this methodology book will provide you with all the necessary
tools for your research into mast cells and basophils, and we wish you every success in the
questofsheddingmorelightonthesefascinatingcelltypes.
Oldenburg,Germany BernhardF.Gibbs
Nottingham,UK FrancoH.Falcone