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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3338-3346
By
From the Department of Allergy and the Department of Experimental
Surgery, National Children's Medical Research Center, Tokyo, Japan;
the Department of Clinical Oncology, The Institute of Medical Science,
University of Tokyo, Tokyo, Japan; and the Department of Bioregulatory
Function, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Human mast cells are derived from CD34+ hematopoietic
cells present in cord blood, bone marrow, and peripheral blood.
However, little is known about the properties of the
CD34+ cells. We demonstrated here that mast cell
progenitors that have distinct phenotypes from other hematopoietic cell
types are present in cord blood by culturing single, sorted
CD34+ cells in 96-well plates or unsorted cells in
methylcellulose. The CD34+ mast cell-committed
progenitors often expressed CD38 and often lacked HLA-DR, whereas
CD34+ erythroid progenitors often expressed both CD38 and
HLA-DR and CD34+ granulocyte-macrophage progenitors often
had CD33 and sometimes expressed CD38. We then cultured single cord
blood-derived CD34+CD38+ cells under
conditions optimal for mast cells and three types of myeloid cells, ie,
basophils, eosinophils, and macrophages. Of 1,200 CD34+CD38+ cells, we were able to detect 13 pure mast cell colonies and 52 pure colonies consisting of either one
of these three myeloid cell types. We found 17 colonies consisting of
two of the three myeloid cell types, whereas only one colony consisted
of mast cells and another cell type. These results indicate that human mast cells develop from progenitors that have unique phenotypes and
that committed mast cell progenitors develop from multipotent hematopoietic cells through a pathway distinct from myeloid lineages including basophils, which have many similarities to mast cells.
MAST CELLS AND BASOPHILS are unique cell
types that possess metachromatic granules composed of highly sulfated
proteoglycans and histamine and that release their granular contents on
cross-linking of their high-affinity receptors for IgE.1
However, mast cells are known to originate from bone marrow progenitors
that migrate into various tissues via blood circulation as immature
cells and undergo complete maturation in the tissues,2,3
whereas basophils complete their maturation within the bone marrow
itself.4 Because of the functional similarities between the
two cell types and the demonstration of human leukemic cells that
possess hybrid granules usually specific for either basophils or mast
cells,5 the possibility that mast cells and basophils share
common bipotent progenitors is still accepted by some investigators. On
the other hand, human basophils were found to possess major basic
protein and Charcot-Leyden crystals, a property they share with
eosinophils, but not mast cells.6 Basophils and eosinophils
also share common progenitors as detected using an in vitro colony
assay,7-9 although these studies cannot completely exclude
the possibility that the common progenitors were multipotent
hematopoietic cells cultured under conditions unsuitable for mast cells.
In contrast to mouse interleukin-3 (IL-3),10,11 which can
act as a mast cell growth factor, human IL-3 by itself does not support
the development of mast cells but instead supports the development of
human basophils and eosinophils.12 The development of human
mast cells was therefore detected in cultures that did not contain
IL-3.13-15 The human mast cell growth factor constitutively expressed on the fibroblast membrane was subsequently cloned and given
several different names, including steel factor (SF), stem cell factor
(SCF), and c-kit ligand.16-18 It is now widely accepted that human mast cells originate from CD34+
cells19,20 and undergo optimal development in the presence of SF.21-23
The primitive human hematopoietic progenitor cells express CD34 but not
CD3824,25 or HLA-DR,26 whereas committed
progenitors, especially erythroid and granulocyte progenitors, often
coexpress CD38 with CD34.23 HLA-DR is often coexpressed on
dendritic cell progenitors,27 B-cell
progenitors,28 erythroid progenitors,29 and
progenitors lacking the ability of stromal cells.30 In the present study, using a single-cell culture system of sorted
CD34+cells, we elucidated the CD34+ progenitors
for human mast cells often coexpressing CD38 and often lacking HLA-DR.
Furthermore, by cultivating singly sorted cord blood-derived
CD34+CD38+ cells under a combined culture
condition suitable for human mast cells,31,32 basophils,
eosinophils, and macrophages,33 we demonstrated that human
mast cells develop from committed progenitors distinct from the three
other myeloid cells.
Cell preparation.
Human umbilical cord blood samples were obtained from normal full-term
deliveries according to the hospital's legal guidelines. Cord blood
was collected in heparinized tubes containing 10 U/mL heparin and
diluted with twice the volume of phosphate-buffered saline (PBS).
Nonphagocytic mononuclear cells were separated by density-gradient
centrifugation using Lymphocyte Separation Medium (LSM; Organon Teknika
Corp, Durham, NC) after depletion of phagocytes with silica (Immuno
Biological Laboratories, Fujioka, Japan). The interface containing
mononuclear cells was collected after density-gradient centrifugation.
Cytokines and antibodies.
Recombinant human thrombopoietin (rhTPO), recombinant human IL-6
(rhIL-6), rhIL-3, and recombinant human erythropoietin (rhEPO) were
generously provided by Kirin Brewery Co, Ltd (Tokyo, Japan). rhSF and
recombinant human granulocyte colony-stimulating factor (rhG-CSF) were
kindly provided by Amgen Biologicals (Thousand Oaks, CA) and Chugai
Pharmaceutical Co (Tokyo, Japan), respectively. The concentrations of
these cytokines used in the first experiments were 100 ng/mL SF, 100 ng/mL IL-6, 40 ng/mL IL-3, 10 ng/mL G-CSF, 2 U/mL EPO, and 4 ng/mL TPO.
Those used in the second series of experiments were 100 ng/mL SF, 50 ng/mL IL-6, and 5 ng/mL rhIL-3 (purchased form Intergen Co, Purchase, NY).
Clone sorting.
Clone sorting was performed using the FACS-Vantage (Becton Dickinson,
Mountain View, CA) equipped with an automated cell deposition unit
(ACDU; Becton Dickinson) using a modified method, as previously described.35 Briefly, cells stained with FITC-labeled
anti-CD34 and either one of PE-labeled anti-CD38, CD33, or HLA-DR and
cells stained only with anti-CD34 were respectively sorted from cord blood mononuclear cells into 96-well flat-bottomed plates (Falcon; Becton Dickinson). As negative controls, cells were stained with FITC-
or PE-conjugated mouse IgG1 (Becton Dickinson).
Cell culture.
In the first series of experiments, the clone-sorted cells were singly
cultured in 96-well plates. Each well contained 200 µL
Immunostaining for tryptase.
Immunostaining for tryptase was performed using a modified method
described by Craig et al.38 The cytospin smears were first air-dried for a few hours at room temperature and then fixed with Carnoy's solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid) for 1 minute. After fixation, the smears were stained for
granular tryptase by the alkaline phosphatase antialkaline phosphatase
(APAAP) method using the Dako APAAP Kit (Dako Corp, Carpinteria, CA)
according to the manufacturer's instructions. Briefly, the smears were
incubated overnight at 4°C with the mouse antihuman tryptase MoAb
(Chemicon, Temecula, CA; diluted to a final concentration of 1 µg/mL
in Tris-HCl-PBS, pH 7.6, + 10% FBS). The smears were then brought to
room temperature and incubated with the Ig fraction of rabbit antiserum
to mouse Igs for 30 minutes. The smears were then incubated with the
alkaline phosphatase mouse antialkaline phosphatase immune complex for
30 minutes. Between each incubation, the smears were rinsed in
Tris-buffered saline (TBS; pH 7.6) for 10 minutes. Finally, the
reaction was developed with the alkaline phosphatase substrate solution
(containing naphthol AS-MX phosphate, Fast Red, and Levamisole) for 20 minutes and then rinsed briefly in a water bath. Negative controls were
performed either by the omission of the primary antibody or by using an isotype-matched mouse IgG1 antibody instead of the primary antibody.
Immunofluorescence staining.
Expression of cell surface antigens on cultured mast cells or basophils
was analyzed by flow cytometry. Briefly, the cells were incubated with
saturating concentrations of the relevant MoAbs for 30 minutes
(4°C), washed, incubated with the FITC-conjugated goat antimouse
IgG+IgM+IgA antibody (30 minutes; Pharmingen), and analyzed by flow
cytometry using a FACScan (Becton Dickinson, San Jose, CA). In each
experiment, negative controls were performed by using isotype-matched
irrelevant control MoAbs.
Phenotypic analysis of mast cell progenitors.
First, we examined the phenotypes of other lineage-committed
hematopoietic progenitors by culturing clone-sorted cells in methylcellulose to compare them with the mast cell-committed
progenitors. As shown in Table 1, most of
the eosinophil progenitors and erythroid progenitors expressed CD34 and
CD38, whereas cord blood cells that gave rise to immature blast cells
in 14 days were mostly CD34+CD38
Morphological analysis of
CD34+CD38+ cell-derived colonies.
In the first series of experiments, we thus demonstrated that mast cell
progenitors preferentially expressed unique phenotypes by culturing
single hematopoietic cells under a condition suitable for mast cells
but not for myeloid cells. In the second part of this study, therefore,
we used a combined culture method that optimally supported at least the
development of mast cells, basophils, eosinophils, and macrophages. We
first cultured the clone-sorted CD34+CD38+
cells in the presence of IL-3, IL-6, and SF for 7 days. The
proliferated cells in each well were then split into 2 parts on day 7 of culture and thereafter cultured either in the presence of SF and
IL-6 for 7 more weeks or in the presence of IL-3 only for 3 more weeks (Fig 1).
Effect of IL-4 on the development of mast cells from
CD34+ or CD34
The present study was designed with the central premise of elucidating
the ontogeny of human mast cells. The CD34+ mast
cell-committed progenitors frequently coexpressed CD38, whereas
primitive CD34+ hematopoietic cells did not express CD38.
The CD38 molecule is expressed on mature granulocytes, some
CD34+ myeloid cells, and lymphocytes at a certain
maturational stage. Mature human mast cells have been
reported to lack CD38 cells.40,41 These results and reports
collectively indicate that the committed progenitors transiently
express this molecule during the development of human mast cells from
multipotent hematopoietic cells. Human CD38 has been recently reported
to be a molecule involved in the regulation of leukocyte adhesion to
endothelial cells,45 indicating that
CD34+CD38+ cells preferably migrate into the
tissues where the appropriate chemoattractants are present, as compared
with CD34+CD38
The authors thank Dr Ryuichi Kaku and the staff of the Kaku Obstetric
Hospital at Hino-shi; Dr Kiyoshi Kawashima; Dr Shigenobu Shoda; and the
staff of the Department of Obstetrics, Gyoda Chuo Hospital for their
continuous support by generously providing the umbilical cord blood. We
also thank Dr Bruce Bochner, Dr Ruby Pawankar, Dr Hidetoshi Kawahara,
Dr Ichiro Nomura, Tomohide Hasegawa, and Hisashi Tomita for their
reviewing of the manuscript, assistance, and advice.
Submitted July 27, 1998; accepted January 11, 1999.
Supported in part by grants to H.S. from the Japanese Ministry of
Health and Welfare (Pediatric Research Grant No. 9-04) and the Japan
Health Sciences Foundation (Grant No. 5114, 1997) and by a grant from
the Japanese Ministry of Education and Culture to T.N.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Hirohisa Saito, MD, PhD, The Department of
Allergy, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan; e-mail:
hsaito{at}nch.go.jp.
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