uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Isolation of transcriptionally active umbilical cord blood-derived basophils expressing FcεRI, HLA-DR and CD203c
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Immunology. (Lars Hellman)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Gunnar Nilsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. (Gunnar Nilsson)
Show others and affiliations
2006 (English)In: Allergy. European Journal of Allergy and Clinical Immunology, ISSN 0105-4538, E-ISSN 1398-9995, Vol. 61, no 9, 1063-1070 p.Article in journal (Refereed) Published
Abstract [en]

Backgrounds: Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil-specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils.

Methods: To obtain transcriptionally active immature basophils, umbilical cord blood cells were cultured in the presence of interleukin (IL)-3. The cells were analysed by flow cytometry and by histological staining.

Results: The continuous presence of IL-3 in cord blood cultures resulted in the expansion of basophil precursors co-expressing Fc epsilon RI and the recently described mast cell/basophil marker, 97A6 (CD203c). Several nonbasophil markers (i.e. CD3, CD14, CD15, CD16, CD19 and CD21) were absent on the cultured basophils. However, we show that in early cultures, almost 60% of the CD203c(+) cells co-express human leukocyte antigen (HLA)-DR, a marker that is absent on mature circulating basophils. The presence of HLA-DR on basophil precursors may explain the low recovery (24 +/- 5.2%) obtained after isolation of cultured basophils, when using a conventional basophil isolation kit that removes HLA-DR+ cells. A novel purification method was developed, including a two-step cocktail of antibodies against selected markers, which resulted in both high purity (95 +/- 0.5%) and recovery (59 +/- 1.5%) of cultured basophils.

Conclusions: We here establish cord blood cultures as a source from which transcriptionally active basophil precursors can be isolated in reasonable quantities for thorough biochemical characterization.

Place, publisher, year, edition, pages
2006. Vol. 61, no 9, 1063-1070 p.
Keyword [en]
basophil, facs analyses, IgE
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97133DOI: 10.1111/j.1398-9995.2006.01149.xISI: 000239514400006PubMedID: 16918508OAI: oai:DiVA.org:uu-97133DiVA: diva2:171936
Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2011-06-29Bibliographically approved
In thesis
1. Hematopoietic Serine Proteases from the Mast Cell Chymase and Tryptase Loci - a Functional and Evolutionary Analysis
Open this publication in new window or tab >>Hematopoietic Serine Proteases from the Mast Cell Chymase and Tryptase Loci - a Functional and Evolutionary Analysis
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish.

Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.

To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.

Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 427
Keyword
Cell and molecular biology, immune system, mast cell, basophil, mast cell chymase locus, multigene tryptase locus, serine protease, evolution, Cell- och molekylärbiologi
Identifiers
urn:nbn:se:uu:diva-8676 (URN)978-91-554-7179-8 (ISBN)
Public defence
2008-05-16, C10:305, BMC, Husargatan 3, Box 596, 75124 Uppsala, 09:15
Opponent
Supervisors
Available from: 2008-04-25 Created: 2008-04-25 Last updated: 2011-07-08Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Authority records BETA

Reimer, JennyHellman, LarsWernersson, Sara

Search in DiVA

By author/editor
Reimer, JennyHellman, LarsWernersson, Sara
By organisation
Molecular ImmunologyDepartment of Cell and Molecular BiologyDepartment of Genetics and Pathology
In the same journal
Allergy. European Journal of Allergy and Clinical Immunology
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 555 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf