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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 901-907
By
From the Department of Veterans Affairs Medical Center and the
Department of Medicine, Medical University of South Carolina,
Charleston, SC; and DNAX Research Institute of Molecular and Cellular
Biology, Palo Alto, CA.
The receptors for interleukin-3 (IL-3), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and IL-5 share a common signaling
subunit © 1998 by The American Society of Hematology.
INTERLEUKIN-3 (IL-3) supports the
development of multiple hematopoietic lineages by interacting with
multipotential and lineage-committed progenitors in
culture.1-3 Studies in our laboratory indicated that IL-3,
as a single factor supports proliferation of the progenitors after they
exit from the cell-cycle dormant state (G0).3,4 IL-3 also
synergizes with IL-6,5 IL-11,6,7 granulocyte colony-stimulating factor (G-CSF),8 leukemia inhibitory
factor,9 thrombopoietin (TPO),10,11 and steel
factor (SF, c-kit ligand)12,13 in triggering cell divisions
of the multipotential progenitors in G0.
In contrast to the positive regulation of myeloid lineages, IL-3 seems
to exert negative effects on the early stages of lymphopoiesis. In our
laboratory, we have established a two-step methylcellulose culture
assay for murine lymphohematopoietic progenitors and characterized their cytokine requirement.14 SF-based cytokine
combinations supported the proliferation and differentiation of
lymphohematopoietic progenitors whereas addition of IL-3 to the
permissive cytokine combinations abrogated the B-lymphoid potential of
the progenitors.15 We subsequently observed that
T-cell16 and natural killer-cell17 potential
of the progenitors is also inhibited by IL-3. These observations raised
the possibility that IL-3 may be a stage-specific negative regulator
and that it may suppress the earliest process of hematopoiesis, ie,
self-renewal of the stem cells. This hypothesis was confirmed later by
our observation that IL-3 abrogates reconstituting ability of
hematopoietic stem cells with long-term engraftment capability.18
Both mouse and human IL-3 receptors are heterodimers consisting of Cytokines.
Purified recombinant murine IL-3 and GM-CSF were purchased from R&D
Systems (Minneapolis, MN). Purified recombinant murine SF was obtained
from Immunex (Seattle, WA). Purified recombinant human IL-6 was a gift
from M. Naruto of Toray Industries (Kamakura, Japan). Purified
recombinant human IL-7 was a gift from Sterling Winthrop Inc
(Collegeville, PA). Purified recombinant human IL-11 was a gift from P. Schendel, Genetics Institute (Cambridge, MA). Purified recombinant
human TPO was prepared by the Cytokine Production Group of Kirin
Brewery (Takasaki, Japan). Recombinant human FLT3/FLK-2 ligand (FL) was
provided by S.D. Lyman of Immunex. Purified recombinant human
erythropoietin (EPO) was provided by the Genetics Institute Clinical
Manufacturing Group (Cambridge, MA). Recombinant human G-CSF was a gift
from A. Shimosaka of Kirin Brewery, Co, Ltd. Unless otherwise
specified, the concentrations of cytokines used were as follows: IL-3,
10 ng/mL; SF, 100 ng/mL; IL-6, 100 ng/mL; IL-7, 200 U/mL; IL-11, 100 ng/mL; TPO, 100 ng/mL; FL, 100 ng/mL; EPO, 2 U/mL; G-CSF, 100 ng/mL;
GM-CSF, 160 ng/mL.
Monoclonal antibodies (MoAbs).
Hybridoma D7 (anti-Ly-6A/E [anti-Sca-1]; rat immunoglobulin G
[IgG]2a) was a gift from P. Kincade of Oklahoma Medical Research Foundation (Oklahoma City, OK). MoAb ACK4 (anti-c-kit; rat IgG2a) was
provided by S.I. Nishikawa of Kyoto University (Kyoto, Japan). Hybridoma RB6-8C5 (anti-mouse granulocytes; rat IgG2b) was provided by
R.L. Coffman of DNAX (Palo Alto, CA). MoAb TER119 (anti-erythrocytes; rat IgG2b) was a gift from T. Kina of Kyoto University. Hybridomas 14.8 (anti-B220; rat IgG2b), M1/70.15.11.5 (anti-macrophages; rat IgG2b),
GK1.5 (anti-CD4; rat IgG2b), and 53-6.72 (anti-CD8; rat IgG2a) were
purchased from American Type Culture Collection (Rockville, MD). 53-2.1 (Biotin-conjugated-anti-Thy-1.2; rat IgG2a), RA3-6B2
(Biotin-conjugated-anti-CD45R/B220; rat IgG2a), RB6-8C5 (Biotin-conjugated-anti-Gr-1; rat IgG2b), and M1/70
(Biotin-conjugated-anti-Mac-1; rat IgG2b) were purchased from
Pharmingen (San Diego, CA). AL1-4A2 (fluorescein isothiocyanate
[FITC]-conjugated anti-Ly-5.2; mouse IgG1) and A20-1.7
(FITC-conjugated anti-Ly-5.1; mouse IgG1) were provided by H. Fleming
of Emory University.
Cell preparations.
Cells from 10- to 15-week-old male and female
Two-step methylcellulose culture for lymphohematopoietic
progenitors.
Four thousand Lin Suspension and clonal cell cultures.
Two hundred Lin In vivo reconstitution experiments.
In studies of knock-out mice, 10- to 12-week-old male C57Bl/6-Ly-5.1
mice were administered with single 850-cGy total-body irradiation via a
4 × 106 V linear accelerator. After irradiation of the
recipient mice, freshly sorted Lin Effects of IL-3 on lymphohematopoietic progenitors.
The results of the studies of lymphohematopoietic progenitors are
presented in Table 1. Regardless of the
origin of the cells, colonies supported by the combination of SF and
IL-11 possessed B-cell potential. Addition of IL-3 to the combination
of SF and IL-11 strongly inhibited the B-cell potential of the primary
colonies of both types of knock-out mice as well as wild-type mice. As we reported previously,14,15 the number of primary colonies was unaffected by IL-3. These results indicated that
Effects of IL-3 on expansion of total cells and progenitors.
Next we studied the effects of IL-3 on the expansion of cells and
colony-forming cells by plating 200 enriched cells in 7-day suspension
culture in the presence of 100 ng/mL SF and 100 ng/mL IL-11 with or
without 100 ng/mL IL-3. As shown in Table
2, the combination of SF and IL-11
increased the total cell counts by 335- to 600-fold, total
colony-forming units (total CFU) by 172- to 402-fold, CFU-GEMM by 26- to 43-fold, and CFU-Meg by 6- to 19-fold, as compared with freshly
enriched cells. Addition of IL-3 resulted in about 30-fold enhancement
of total cell counts and several-fold increase in the total CFU,
CFU-GEMM, and CFU-Meg. These results confirmed that the
myelostimulatory effects of IL-3 are transduced by both
Effects of IL-3 on long-term repopulating cells (LTRC).
We then tested the in vivo reconstituting ability of the cultured
cells. The suspension cultures were initiated with 200 Lin
Effects of GM-CSF.
The observation that the
There is significant current interest in hematology/oncology fields
regarding in vitro expansion of hematopoietic stem cells and
progenitors.36-51 A number of investigators have already
shown that it is possible to increase the number of hematopoietic
progenitors in culture by using combinations of early-acting
cytokines.36-51 Because of the well-known myelopoietic
effects of IL-3, the majority of preclinical protocols for
murine37-43 and human36,44-51 cells included
IL-3. We previously noted negative effects of IL-3 on the ability of
cultured cells to engraft the marrow of recipient mice.18
Our observation was in agreement with the report from Peters et
al52 that suspension culture of murine marrow cells in the
presence of IL-3, IL-6, IL-11, and SF results in impairment of the
engrafting capability of the cultured cells.
Submitted November 24, 1997;
accepted April 1, 1998.
We thank Dr Haiqun Zeng for assistance in cell sorting; Dr Pamela N. Pharr and Anne G. Leary for assistance in preparation of this
manuscript; and the staff of Radiation Oncology Department of the
Medical University of South Carolina for assistance in irradiation of
mice.
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