Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors

M. W. Al-Rabia, M. G. Blaylock, D. W. Sexton, L. Thomson, G. M. Walsh

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions.

Objectives The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro .

Methods Purified human peripheral blood CD34(+) cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2(TM) light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation.

Results Positive immunostaining for MBP and ECP was detectable in a proportion (15-20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of similar to 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE.

Conclusion These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

Original languageEnglish
Pages (from-to)640-648
Number of pages8
JournalClinical & experimental allergy
Volume33
Issue number5
DOIs
Publication statusPublished - May 2003

Keywords

  • CD34(+) progenitors
  • ECP
  • eosinophilopoiesis
  • eosinophils
  • MBP
  • membrane receptor phenotype
  • recombinant human interleukin-5
  • mononuclear cell-cultures
  • differentiating eosinophils
  • asthmatic subjects
  • alpha-subunit
  • expression
  • maturation
  • commitment
  • develop
  • lineage

Cite this

Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors. / Al-Rabia, M. W. ; Blaylock, M. G.; Sexton, D. W.; Thomson, L. ; Walsh, G. M.

In: Clinical & experimental allergy, Vol. 33, No. 5, 05.2003, p. 640-648.

Research output: Contribution to journalArticle

Al-Rabia, M. W. ; Blaylock, M. G. ; Sexton, D. W. ; Thomson, L. ; Walsh, G. M. / Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors. In: Clinical & experimental allergy. 2003 ; Vol. 33, No. 5. pp. 640-648.
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abstract = "Background Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions.Objectives The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro .Methods Purified human peripheral blood CD34(+) cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2(TM) light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation.Results Positive immunostaining for MBP and ECP was detectable in a proportion (15-20{\%}) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of similar to 80{\%} by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE.Conclusion These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.",
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T1 - Granule protein changes and membrane receptor phenotype in maturing human eosinophils cultured from CD34+ progenitors

AU - Al-Rabia, M. W.

AU - Blaylock, M. G.

AU - Sexton, D. W.

AU - Thomson, L.

AU - Walsh, G. M.

PY - 2003/5

Y1 - 2003/5

N2 - Background Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions.Objectives The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro .Methods Purified human peripheral blood CD34(+) cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2(TM) light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation.Results Positive immunostaining for MBP and ECP was detectable in a proportion (15-20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of similar to 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE.Conclusion These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

AB - Background Eosinophils are now recognized as major effector cells in allergic and asthmatic disease with a potent armoury of mediators whose release makes a major contribution to the inflammation underlying these conditions.Objectives The purpose of this study was to compare cultured eosinophils (CE) with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and to analyse the expression and storage of the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP) during eosinophil maturation in vitro .Methods Purified human peripheral blood CD34(+) cells were cultured in the presence of recombinant human IL-3, IL-5, rhGM-CSF, SCF, and FLT-3 ligand. PBE were isolated by density gradient centrifugation and negative immunomagnetic selection. Expression of CD11b, CD18, CD45, CD45RA, CD45RB, CD45RO, CD69, CD95, IL-5Ralpha, IL-9Ralpha, CCR1, CCR3, and CXCR4 by CE as they matured in culture were assessed by immunostaining and flow cytometry and expression of these receptors compared with freshly isolated PBE. Immunohistochemical staining and labophot-2(TM) light microscopy determined expression of MBP, ECP, and CD69 during eosinophil maturation.Results Positive immunostaining for MBP and ECP was detectable in a proportion (15-20%) of CE as early as 3 days of culture even though these cells were mononuclear in appearance. The numbers of CE positive for both granule proteins increased in rhIL-3 and rhIL-5 treated cells to a maximum of similar to 80% by day 28. Maturing eosinophils exhibited positive immunostaining for CD69 after 14, 21 and 28 days of culture. Compared with PBE, CE had lower expression of pan-CD45 and CD45 isoforms, CD95 and CD11b. In contrast, the specific mean fluorescence for CD69, CD18, IL-5Ralpha, and IL-9Ralpha was significantly elevated for CE compared with PBE. CCR3 expression by CE and PBE was similar with no expression of CXCR4 detected by either CE or PBE. No significant difference in expression of CCR1 was found between CE and PBE.Conclusion These data suggest that CE and PBE share many phenotypic properties and both MBP and ECP appear early in eosinophil development in vitro. However, there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

KW - CD34(+) progenitors

KW - ECP

KW - eosinophilopoiesis

KW - eosinophils

KW - MBP

KW - membrane receptor phenotype

KW - recombinant human interleukin-5

KW - mononuclear cell-cultures

KW - differentiating eosinophils

KW - asthmatic subjects

KW - alpha-subunit

KW - expression

KW - maturation

KW - commitment

KW - develop

KW - lineage

U2 - 10.1046/j.1365-2222.2003.01639.x

DO - 10.1046/j.1365-2222.2003.01639.x

M3 - Article

VL - 33

SP - 640

EP - 648

JO - Clinical & experimental allergy

JF - Clinical & experimental allergy

SN - 0954-7894

IS - 5

ER -